Erythrocyte Production and Destruction 8

Kathryn Doig

OUTLINE OBJECTIVES
Normoblastic Maturation After completion of this chapter, the reader will be able to:
Terminology
Maturation Process 1. List and describe the erythroid precursors in order of 1 0. Define and differentiate erythron and RBC mass.
Criteria Used in Identification maturity, including the morphologic characteristics, 11. Explain how hypoxia stimulates RBC production.
of the Erythroid Precursors cellular activities, normal location, and length of time 12. Describe the general chemical composition of eryth-
Maturation Sequence in the stage for each. ropoietin (EPO) and name the site of production.
Erythrokinetics 2. Correlate the erythroblast, normoblast, and rubriblast 13. Discuss the various mechanisms by which EPO
Hypoxia— the Stimulus to nomenclatures for red blood cell (RBC) stages. contributes to erythropoiesis.
Red Blood Cell Production 3. Name the stage of erythroid development when given 14. Define and explain apoptosis resulting from Fas/FasL
Other Stimuli to a written description of the morphology of a cell in a interactions and how this regulatory mechanism
Erythropoiesis Wright-stained bone marrow preparation. applies to erythropoiesis.
Microenvironment of the
4. List and compare the cellular organelles of immature 15. Explain the effect of Bcl-XL (Bcl-2 like protein 1) and
Bone Marrow
and mature erythrocytes and describe their specific the general mechanism by which it is stimulated in
Erythrocyte Destruction
Macrophage-Mediated functions. red blood cell progenitors.
Hemolysis (Extravascular 5. Name the erythrocyte progenitors and distinguish 16. Describe the features of the bone marrow that con-
Hemolysis) them from precursors. tribute to establishing the microenvironment neces-
Mechanical Hemolysis 6. Explain the nucleus-to-cytoplasm (N:C) ratio, de- sary for the proliferation of RBCs, including location
(Intravascular Hemolysis) scribe the appearance of a cell when given the N:C and arrangement relative to other cells, with particu-
ratio, and estimate the N:C ratio from the appearance lar emphasis on the role of fibronectin.
of a cell. 17. Discuss the role of macrophages in RBC develop-
7. Explain how reticulocytes can be recognized in a ment.
Wright-stained peripheral blood film. 18. Explain how RBCs enter the bloodstream and how
8. Define and differentiate the terms polychromasia, dif- premature entry is prevented and, when appropriate,
fuse basophilia, punctate basophilia, and basophilic promoted.
stippling. 19. Describe the characteristics of senescent RBCs and
9. Discuss the differences between the reticulum of explain why RBCs age.
reticulocytes and punctate basophilic stippling in 20. Explain and differentiate the two normal mechanisms of
composition and conditions for microscopic viewing. erythrocyte destruction, including location and process.

CASE STUDY
After studying the material in this chapter, the reader should be able to respond to the following case study:

A 42-year-old premenopausal woman has emphysema. oxygen therapy for several months, her RBC count dropped
This lung disease impairs the ability to oxygenate the to 5.0 3 1012/L.
blood, so patients experience significant fatigue and short- 1. What physiologic response explains the elevation of
ness of breath. To alleviate these symptoms, oxygen is the first RBC count?
typically prescribed, and this patient has a portable oxy- 2. What hormone is responsible? How is its production
gen tank she carries with her at all times, breathing stimulated? What is the major way in which it acts?
through nasal cannulae. Before she began using oxygen, 3. What explains the decline in RBC count with oxygen
her red blood cell (RBC) count was 5.8 3 1012/L. After therapy for this patient?

95
96 PART II  Blood Cell Production, Structure, and Function

T
he red blood cell (RBC), or erythrocyte, provides a clas- Maturation Process
sic example of the biological principle that cells have Erythroid Progenitors
specialized functions and that their structures are As described in Chapter 7, the morphologically identifiable
specific for those functions. The erythrocyte has one true erythrocyte precursors develop from two functionally iden-
function: to carry oxygen from the lung to the tissues, where tifiable progenitors, burst-forming unit–erythroid (BFU-E)
the oxygen is released. This is accomplished by the attachment and colony-forming unit–erythroid (CFU-E), both commit-
of the oxygen to hemoglobin (HGB), the major cytoplasmic ted to the erythroid cell line. Estimates of time spent at each
component of mature RBCs. The role of the RBC in returning stage suggest that it takes about one week for the BFU-E to
carbon dioxide to the lungs and buffering the pH of the blood mature to the CFU-E and another week for the CFU-E to
is important but is quite secondary to its oxygen-carrying become a pronormoblast,1 which is the first morphologi-
function. To protect this essential life function, the mecha- cally identifiable RBC precursor. While at the CFU-E stage,
nisms controlling development, production, and normal the cell completes approximately three to five divisions
destruction of RBCs are fine-tuned to avoid interruptions in before maturing further.1 As seen later, it takes approxi-
oxygen delivery, even under adverse conditions such as blood mately another 6 to 7 days for the precursors to become
loss with hemorrhage. This chapter and subsequent chapters mature enough to enter the circulation, so approximately 18
discussing iron, RBC metabolism, membrane structure, and to 21 days are required to produce a mature RBC from
hemoglobin constitute the foundation for understanding the the BFU-E.
body’s response to diminished oxygen-carrying capacity of the
blood, called anemia. Erythroid Precursors
The mammalian erythrocyte is unique among animal cells Normoblastic proliferation, similar to the proliferation of
in having no nucleus in its mature, functional state. While am- other cell lines, is a process encompassing replication (i.e.,
phibians and birds possess RBCs, their nonmammalian RBCs division) to increase cell numbers and development from
retain the nuclei throughout the cells’ lives. The implications of immature to mature cell stages (Figure 8-1). The earliest
this unique mammalian adaptation are significant for cell morphologically recognizable erythrocyte precursor, the pro-
function and life span. normoblast, is derived via the BFU-E and CFU-E from the
pluripotential stem cells, as discussed in Chapter 7. The pro-
normoblast is able to divide, with each daughter cell maturing
NORMOBLASTIC MATURATION
to the next stage of development, the basophilic normoblast.
Terminology Each of these cells can divide, with each of its daughter cells
RBCs are formally called erythrocytes. The nucleated precursors maturing to the next stage, the polychromatic normoblast.
in the bone marrow are called erythroblasts. They also may be Each of these cells also can divide and mature. In the erythro-
called normoblasts, which refers to developing nucleated cells cyte cell line, there are typically three and occasionally as
(i.e., blasts) with normal appearance. This is in contrast to the many as five divisions2 with subsequent nuclear and cytoplas-
abnormal appearance of the developing nucleated cells in mic maturation of the daughter cells, so from a single pronor-
megaloblastic anemia, in which the erythroblasts are called moblast, 8 to 32 mature RBCs usually result. The conditions
megaloblasts because of their large size. under which the number of divisions can be increased or re-
Three nomenclatures are used for naming the erythroid duced are discussed later.
precursors (Table 8-1). The erythroblast terminology is used The cellular activities at each stage of development
primarily in Europe. Like the normoblastic terminology used described below occur in an orderly and sequential process.
more often in the United States, it has the advantage of being It is often likened to a computer program that once acti-
descriptive of the appearance of the cells. Some prefer the vated runs certain processes in a specified order at specified
rubriblast terminology because it parallels the nomenclature times. The details of the developmental program are becom-
used for granulocyte development (Chapter 12). Normoblastic ing clearer, and selected details are provided in these
terminology is used in this chapter. descriptions.

TABLE 8-1  ​Three Erythroid Precursor Nomenclature Systems

Normoblastic Rubriblastic Erythroblastic
Pronormoblast Rubriblast Proerythroblast
Basophilic normoblast Prorubricyte Basophilic erythroblast
Polychromatic (polychromatophilic) normoblast Rubricyte Polychromatic (polychromatophilic) erythroblast
Orthochromic normoblast Metarubricyte Orthochromic erythroblast
Polychromatic (polychromatophilic) erythrocyte* Polychromatic (polychromatophilic) Polychromatic (polychromatophilic)
erythrocyte* erythrocyte*
Erythrocyte Erythrocyte Erythrocyte

*Polychromatic erythrocytes are called reticulocytes when observed with vital stains.
 CHAPTER 8  Erythrocyte Production and Destruction 97

CFU-E

Pronormoblast

Basophilic
normoblast

Polychromatophilic
normoblast

Orthochromic
normoblast

Reticulocyte – BM

Reticulocyte – PB

Erythrocyte

Figure 8-1  ​Typical production of erythrocytes from two pronormoblasts illustrating three mitotic divisions among precursors. BM, Bone marrow; CFU, colony
forming unit–erythroid; PB, peripheral blood.

Criteria Used in Identification of the Erythroid parachromatin. This chromatin/parachromatin distinction is

Precursors more dramatic than in other cell lines. Ultimately, the nucleus
Morphologic identification of blood cells depends on a well- becomes quite condensed, with no parachromatin evident at
stained peripheral blood film or bone marrow smear all, and the nucleus is said to be pyknotic.
(Chapters 16 and 17). In hematology, a modified Romanowsky 4. Nucleoli disappear. Nucleoli represent areas where the
stain, such as Wright or Wright-Giemsa, is commonly used. The ribosomes are formed and are seen early in cell develop-
descriptions that follow are based on the use of these types of ment as cells begin actively synthesizing proteins. As RBCs
stains. mature, the nucleoli disappear, which precedes the ultimate
The stage of maturation of any blood cell is determined cessation of protein synthesis.
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, BOX 8-1  ​Nucleus-to-Cytoplasm (N:C) Ratio
homogeneity), nuclear diameter, nucleus:cytoplasm (N:C)
ratio (Box 8-1), presence or absence of nucleoli, and cyto- The nucleus-to-cytoplasm (N:C) ratio is a morphologic feature used
plasmic color. to identify and stage red blood cell and white blood cell precursors.
As RBCs mature, several general trends affect their appear- The ratio is a visual estimate of what area of the cell is occupied by
ance. Figure 8-2 graphically represents these trends. the nucleus compared with the cytoplasm. If the areas of each are
1. The overall diameter of the cell decreases. approximately equal, the N:C ratio is 1:1. Although not mathemati-
2. The diameter of the nucleus decreases more rapidly than cally proper, it is common for ratios other than 1:1 to be referred to
does the size of the cell. As a result, the N:C ratio also de- as if they were fractions. If the nucleus takes up less than 50% of the
creases. area of the cell, the proportion of nucleus is lower, and the ratio is
3. The nuclear chromatin pattern becomes coarser, clumped, lower (e.g., 1:5 or less than 1). If the nucleus takes up more than
and condensed. The nuclear chromatin of RBCs is inherently 50% of the area of the cell, the ratio is higher (e.g., 3:1 or 3). In the
coarser than that of myeloid precursors. It becomes even red blood cell line, the proportion of nucleus shrinks as the cell ma-
coarser and more clumped as the cell matures, developing a tures and the cytoplasm increases proportionately, although the
raspberry-like appearance, in which the dark staining of the overall cell diameter grows smaller. In short, the N:C ratio decreases.
chromatin is distinct from the almost white appearance of the
98 PART II  Blood Cell Production, Structure, and Function

A B C D
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 a very dark purple-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.)

5. The cytoplasm changes from blue to gray-blue to salmon ultimately ends with a thoroughly salmon pink color when
pink. Blueness or basophilia is due to acidic components that the ribosomes are gone and only hemoglobin remains.
attract the basic stain, such as methylene blue. The
degree of cytoplasmic basophilia correlates with the amount Maturation Sequence
of ribosomal RNA. These organelles decline over the life of Table 8-2 lists the stages of RBC development in order and
the developing RBC, and the blueness fades. Pinkness called provides a convenient comparison. The listing makes it
eosinophilia or acidophilia is due to accumulation of more ba- appear that these stages are clearly distinct and easily identifi-
sic components that attract the acid stain eosin. Eosinophilia able. The process of cell maturation is a gradual process, with
of erythrocyte cytoplasm correlates with the accumulation of changes occurring in a generally predictable sequence but
hemoglobin as the cell matures. Thus the cell starts out being with some variation for each individual cell. The identifica-
active in protein production on the ribosomes that make the tion of a given cell’s stage depends on the preponderance of
cytoplasm basophilic, transitions through a period in which characteristics, although the cell may not possess all the
the red of hemoglobin begins to mix with that blue, and features of the archetypal descriptions that follow. Essential

TABLE 8-2  ​Normoblastic Series: Summary of Stage Morphology

Nucleus-to- Bone Marrow
Cell or Stage Diameter Cytoplasm Ratio Nucleoli % in Bone Marrow Transit Time
Pronormoblast 12–20 mm 8:1 1–2 1% 24 hr
Basophilic normoblast 10–15 mm 6:1 0–1 1%–4% 24 hr
Polychromatic normoblast 10–12 mm 4:1 0 10%–20% 30 hr
Orthochromic normoblast 8–10 mm 1:2 0 5%–10% 48 hr
Bone marrow polychromatic 8–10 mm No nucleus 0 1% 24–48 hr
erythrocyte*

*Also called reticulocyte.

 CHAPTER 8  Erythrocyte Production and Destruction 99

features of each stage are in italics in the following descrip- Location.  ​The pronormoblast is present only in the bone
tions. The cellular functions described subsequently also are marrow in healthy states.
summarized in Figure 8-3.
Cellular Activity.  ​The pronormoblast begins to accu-
Pronormoblast (Rubriblast) mulate the components necessary for hemoglobin produc-
Figure 8-4 shows the pronormoblast. tion. The proteins and enzymes necessary for iron uptake and
protoporphyrin synthesis are produced. Globin production
Nucleus.  ​The nucleus takes up much of the cell (N:C begins.3
ratio of 8:1). The nucleus is round to oval, containing one or two
nucleoli. The purple red chromatin is open and contains few, if any, Length of Time in This Stage.  ​This stage lasts slightly
fine clumps. more than 24 hours.3

Cytoplasm.  ​The cytoplasm is dark blue because of the Basophilic Normoblast (Prorubricyte)
concentration of ribosomes. The Golgi complex may be visible Figure 8-5 shows the basophilic normoblast.
next to the nucleus as a pale, unstained area. Pronormoblasts
may show small tufts of irregular cytoplasm along the periph- Nucleus.  ​The chromatin begins to condense, revealing
ery of the membrane. clumps along the periphery of the nuclear membrane and a
few in the interior. As the chromatin condenses, the parachro-
Division. The pronormoblast undergoes mitosis and gives matin areas become larger and sharper, and the N:C ratio
rise to two daughter pronormoblasts. More than one division decreases to about 6:1. The chromatin stains deep purple-red.
is possible before maturation into basophilic normoblasts. Nucleoli may be present early in the stage but disappear later.
tahir99

48 hr 30 hr 48 hr 48-72 hr
Pro- Basophilic Poly- Ortho- Reticulo-
normoblast normoblast chromatic chromic cyte
normoblast normoblast

Average 12
cell 10
A diameter
(m) 8
6
RNA
Rate of content
B RNA
synthesis

Rate of
DNA
C DNA
content
synthesis

34 Total protein
Hemoglobin concentration/cell
concentration 24
D (pg) 14
4 Acidophilia

Bone marrow
Figure 8-3  ​Changes in cellular diameter, RNA synthesis and content, DNA synthesis and content, protein and hemoglobin content during red blood cell
development. A, Red blood cell diameter (solid line) shrinks from the pronormoblast to the reticulocyte stage. B, The rate of RNA synthesis (solid line) for
protein production is at its peak at the pronormoblast stage and ends in the orthochromic normoblast stage. The RNA accumulates so that the RNA content
(dashed line) remains relatively constant into the orthochromic normoblast stage when it begins to degrade, being eliminated by the end of the reticulocyte
stage. C, The rate of DNA synthesis (solid line) correlates to those stages of development that are able to divide; the pronormoblast, basophilic normoblast,
and early polychromatic normoblast stages. DNA content (dashed line) of a given cell remains relatively constant until the nucleus begins to break up and be
extruded during the orthochromic normoblast stage. There is no DNA, i.e., no nucleus, in reticulocytes. D, The dashed line represents the total protein con-
centration which declines slightly during maturation. Proteins other than hemoglobin predominate in early stages. The hemoglobin concentration (solid line)
begins to rise in the basophilic normoblast stage, reaching its peak in reticulocytes and representing most of the protein in more mature cells. Hemoglobin
synthesis is visible as acidophilia (dotted line) that parallels hemoglobin accumulation but is delayed since the earliest production of hemoglobin in basophilic
normoblasts is not visible microscopically. (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.)
100 PART II  Blood Cell Production, Structure, and Function

A B
Figure 8-4  ​A, Pronormoblast (rubriblast), bone marrow (Wright stain, 31000). B, Electron micrograph of pronormoblast (315,575). (B from Rodak BF,
Carr JH: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders, an imprint of Elsevier Inc.)

A B
Figure 8-5  ​A, Basophilic normoblast (prorubricyte), bone marrow (Wright stain, 31000). B, Electron micrograph of basophilic normoblast (315,575).
(B from Rodak BF, Carr JH: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders, an imprint of Elsevier Inc.)

Cytoplasm.  ​When stained, the cytoplasm may be a deeper, Polychromatic (Polychromatophilic) Normoblast
richer blue than in the pronormoblast—hence the name (Rubricyte)
basophilic for this stage. Figure 8-6 shows the polychromatic normoblast.

Division.  ​The basophilic normoblast undergoes mitosis, giv- Nucleus.  ​The chromatin pattern varies during this stage
ing rise to two daughter cells. More than one division is possible of development, showing some openness early in the stage but
before the daughter cells mature into polychromatic normoblasts. becoming condensed by the end. The condensation of chromatin
reduces the diameter of the nucleus considerably, so the N:C
Location.  ​The basophilic normoblast is present only in ratio decreases from 4:1 to about 1:1 by the end of the stage.
the bone marrow in healthy states. Notably, no nucleoli are present.

Cellular Activity.  ​Detectable hemoglobin synthesis oc- Cytoplasm.  ​This is the first stage in which the pink color
curs,3 but the many cytoplasmic organelles, including ribosomes associated with stained hemoglobin can be seen. The stained
and a substantial amount of messenger ribonucleic acid (RNA; color reflects the accumulation of hemoglobin pigmentation
chiefly for hemoglobin production), completely mask the over time and concurrent decreasing amounts of RNA. The
minute amount of hemoglobin pigmentation. color produced is a mixture of pink and blue, resulting in a
murky gray-blue. The stage’s name refers to this combination of
Length of Time in This Stage.  ​This stage lasts slightly multiple colors, because polychromatophilic means “many color
more than 24 hours.3 loving.”
 CHAPTER 8  Erythrocyte Production and Destruction 101

A B
Figure 8-6  ​A, Polychromatic normoblast (rubricyte), bone marrow (Wright stain, ×1000). B, Electron micrograph of polychromatic normoblast (×15,575).
(B from Rodak BF, Carr JH: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders, an imprint of Elsevier Inc.)

Division.  ​This is the last stage in which the cell is capable Orthochromic Normoblast (Metarubricyte)
of undergoing mitosis, although likely only early in the stage. Figure 8-7 shows the orthochromic normoblast.
The polychromatic normoblast goes through mitosis, produc-
ing daughter cells that mature and develop into orthochromic Nucleus.  ​The nucleus is completely condensed (i.e., pyk-
normoblasts. notic) or nearly so. As a result, the N:C ratio is low or
approximately 1:2.
Location.  ​The polychromatic normoblast is present only
in the bone marrow in healthy states. Cytoplasm.  ​The increase in the salmon-pink color of the
cytoplasm reflects nearly complete hemoglobin production.
Cellular Activity.  ​Hemoglobin synthesis increases, and the The residual ribosomes react with the basic component of
accumulation begins to be visible in the color of the cytoplasm. the stain and contribute a slightly bluish hue to the cell, but
Cellular organelles are still present, particularly ribosomes, that fades toward the end of the stage as the organelles are
which contribute a blue aspect to the cytoplasm. The progressive degraded.
condensation of the nucleus and disappearance of nucleoli are
evidence of progressive decline in transcription of deoxyribo- Division.  ​The orthochromic normoblast is not capable of
nucleic acid (DNA). division due to the condensation of the chromatin.

Length of Time in This Stage.  ​This stage lasts approxi- Location.  ​The orthochromic normoblast is present only
mately 30 hours.3 in the bone marrow in healthy states.

A B
Figure 8-7  ​A, Orthochromic normoblast (metarubricyte), bone marrow (Wright stain, 31000). B, Electron micrograph of orthochromic normoblast
(320,125). (B from Rodak BF, Carr JH: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders, an imprint of Elsevier Inc.)
102 PART II  Blood Cell Production, Structure, and Function

Cellular Activity.  ​Hemoglobin production continues Cytoplasm.  ​The cytoplasm can be compared with that of
on the remaining ribosomes using messenger RNA produced the late orthochromic normoblast in that the predominant
earlier. Late in this stage, the nucleus is ejected from the cell. color is that of hemoglobin. By the end of the polychromatic
The nucleus moves to the cell membrane and into a erythrocyte stage, the cell is the same color as a mature RBC,
pseudopod-like projection. As part of the maturation salmon pink. It remains larger than a mature cell, however. The
program, loss of vimentin, a protein responsible for holding shape of the cell is not the mature biconcave disc but is irregu-
organelles in proper location in the cytoplasm, is probably lar in electron micrographs (Figure 8-8, B).
important in the movement of the nucleus to the cell periph-
ery.1 Ultimately, the nucleus-containing projection separates Division.  ​Lacking a nucleus, the polychromatic erythro-
from the cell by having the membrane seal and pinch off the cyte cannot divide.
projection with the nucleus enveloped by cell membrane.4
Nonmuscle myosin of the membrane is important in this Location.  ​The polychromatic erythrocyte resides in the
pinching process.5 The enveloped extruded nucleus, called a bone marrow for 1 day or longer and then moves into the
pyrenocyte,1 is then engulfed by bone marrow macrophages. The peripheral blood for about 1 day before reaching maturity.
macrophages recognize phosphatidlyserine on the pyreno- During the first several days after exiting the marrow, the
cyte surface as an “eat me” flag.6 Other organelles are polychromatic erythrocyte is retained in the spleen for
extruded and ingested in similar fashion. Often, small pitting of inclusions and membrane polishing by splenic
fragments of nucleus are left behind if the projection is macrophages, which results in the biconcave discoid
pinched off before the entire nucleus is enveloped. These mature RBC.7
fragments are called Howell-Jolly bodies when seen in periph-
eral blood cells (Table 19-3 and Figure 19-1) and are typically Cellular Activity.  ​The polychromatic erythrocyte com-
removed from the cells by the splenic macrophage pitting pletes production of hemoglobin from residual messenger
process once the cell enters the circulation. RNA using the remaining ribosomes. The cytoplasmic protein
production machinery is simultaneously being dismantled.
Length of Time in This Stage.  ​This stage lasts approxi- Endoribonuclease, in particular, digests the ribosomes. The
mately 48 hours.3 acidic components that attract the basophilic stain decline
during this stage to the point that the polychromatophilia is
Polychromatic (Polychromatophilic) not readily evident in the polychromatic erythrocytes on a
Erythrocyte or Reticulocyte normal peripheral blood film stained with Wright stain. A
Figure 8-8 shows the polychromatic erythrocyte. small amount of residual ribosomal RNA is present, however,
and can be visualized with a vital stain such as new methylene
Nucleus.  ​Beginning at the polychromatic erythrocyte blue, so called because the cells are stained while alive in
stage, there is no nucleus. The polychromatic erythrocyte is a suspension (i.e., vital), before the film is made (Box 8-2). The
good example of the prior statement that a cell may not have residual ribosomes appear as a mesh of small blue strands, a
all the classic features described but may be staged by the reticulum, or, when more fully digested, merely blue dots
preponderance of features. In particular, when a cell loses its (Figure 8-9). When so stained, the polychromatic erythrocyte
nucleus, regardless of cytoplasmic appearance, it is a poly- is called a reticulocyte. However, the name reticulocyte is often
chromatic erythrocyte. used to refer to the stage immediately preceding the mature

A B
Figure 8-8  ​A, Polychromatic erythrocyte (shift reticulocyte), peripheral blood (Wright stain, 31000). B, Scanning electron micrograph of polychromatic
erythrocyte (35000). (B from Rodak BF, Carr JH: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders, an imprint of Elsevier Inc.)
 CHAPTER 8  Erythrocyte Production and Destruction 103

Erythrocyte
BOX 8-2  ​Cellular Basophilia: Diffuse and Punctate Figure 8-10 shows the erythrocyte.

The reticulum of a polychromatic erythrocyte (reticulocyte) is not Nucleus.  ​No nucleus is present in mature RBCs.
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 Cytoplasm.  ​The mature circulating erythrocyte is a bi-
appearance, the reticulocyte is called a polychromatic erythrocyte concave disc measuring 7 to 8 mm in diameter, with a thickness
because it lacks a nucleus and is no longer an erythroblast but has of about 1.5 to 2.5 mm. On a stained blood film, it appears as
a bluish tinge. When polychromatic erythrocytes are prominent on a a salmon pink-staining cell with a central pale area that corre-
peripheral blood film, the examiner uses the comment polychromasia sponds to the concavity. The central pallor is about one third
or polychromatophilia. Wright-stained polychromatic erythrocytes are the diameter of the cell.
also called diffusely basophilic erythrocytes for their regular bluish
tinge. This term distinguishes polychromatic erythrocytes from red Division.  ​The erythrocyte cannot divide.
blood cells with punctate basophilia, in which the blue appears in
distinct dots throughout the cytoplasm. More commonly known as Location and Length of Time in This Stage.  ​Mature
basophilic stippling (Table 19-3 and Figure 19-1), punctate baso- RBCs remain active in the circulation for approximately 120
philia is associated with some anemias. Similar to the basophilia of days.11 Aging leads to their removal by the spleen as described
polychromatic erythrocytes, punctate basophilia is due to residual subsequently.
ribosomal RNA, but the RNA is degenerate and stains deeply with
Wright stain. 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. It has a surface-to-volume ra-
tio and shape that enable optimal gas exchange to occur. If the
cell were to be spherical, it would have hemoglobin at the cen-
ter of the cell that would be relatively distant from the mem-
brane 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—
that is, able to flex but return to its original shape. The interac-
tion of various membrane components described in Chapter 9
creates these properties. RBCs must squeeze through small
spaces such as the basement membrane of the bone 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. Deformability is crucial for RBCs to enter
and subsequently remain in the circulation.

Figure 8-9  ​Reticulocytes at arrows, peripheral blood (new methylene blue ERYTHROKINETICS
stain, 31000).
Erythrokinetics is the term describing the dynamics of RBC
production and destruction. To understand erythrokinetics, it
erythrocyte, even when stained with Wright stain and without is helpful to appreciate the concept of the erythron. Erythron is
demonstrating the reticulum. the name given to the collection of all stages of erythrocytes
A second functional change in polychromatic erythrocytes is throughout the body: the developing precursors in the bone
the reduced production of receptors for the adhesive molecules marrow and the circulating erythrocytes in the peripheral
that hold developing RBCs in the marrow (see details later).8-10 blood and the vascular spaces within specific organs such as
As these receptors decline, cells are freed to leave the marrow. the spleen. When the term erythron is used, it conveys the
concept of a unified functional tissue. The erythron is distin-
Length of Time in This Stage.  ​The cell typically guished from the RBC mass. The erythron is the entirety of
remains a polychromatic erythrocyte for about 3 days,3 with erythroid cells in the body, whereas the RBC mass refers only
the first 2 days spent in the marrow and the third spent in the to the cells in circulation. This discussion of erythrokinetics
peripheral blood, although possibly sequestered in the spleen. begins by looking at the erythrocytes in the bone marrow and
104 PART II  Blood Cell Production, Structure, and Function

A B
Figure 8-10  ​A, Mature erythrocytes and one lymphocyte, peripheral blood (Wright stain, 31000). B, Scanning electron micrograph of mature erythrocytes.
(A from Rodak BF, Carr JH. Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders, an imprint of Elsevier Inc.)

the factors that affect their numbers, their progressive develop-

BOX 8-3  ​Hypoxia and Red Blood Cell Production
ment, and their ultimate release into the blood.
Teleologically speaking, the location of the body’s hypoxia sensor in
Hypoxia—the Stimulus to Red Blood Cell the kidney is practical,1 because the kidney receives approximately
Production 20% of the cardiac output2 with little loss of oxygen from the levels
As mentioned previously, the role of RBCs is to carry oxygen. leaving the heart. The location provides early detection when oxygen
To regulate the production of RBCs for that purpose, the body levels decline. Making the hypoxia sensor the cell that is able to
requires a mechanism for sensing whether there is adequate stimulate red blood cell (RBC) production also is practical, because
oxygen being carried to the tissues. If not, RBC production regardless of the cause of hypoxia, having more RBCs should help to
and the functional efficiency of existing cells must be overcome it. The hypoxia might result from decreased RBC numbers,
enhanced. Thus a second feature of the oxygen-sensing system as with hemorrhage.
must be a mechanism for influencing the production of RBCs. Decreased RBC number, however, is only one cause of hy-
The primary oxygen-sensing system of the body is located in poxia. Another cause is the failure of each RBC to carry as much
peritubular fibroblasts of the kidney.12-14 Hypoxia, too little tis- oxygen as it should. This can occur because the hemoglobin is
sue oxygen, is detected by the peritubular cells, which produce defective or because there is not enough hemoglobin in each cell.
erythropoietin (EPO), the major stimulatory cytokine for RBCs. The hypoxia may be unrelated to the RBCs in any way; poor lung
Under normal circumstances, the amount of EPO produced function resulting in diminished oxygenation of existing RBCs is
fluctuates very little, maintaining a level of RBC production that an example.
is sufficient to replace the approximately 1% of RBCs that The kidney’s hypoxia sensor cannot know why there is hy-
normally die each day (see section on erythrocyte destruction). poxia, but it does not matter. Even when there are plenty of RBCs
When there is hemorrhage, increased RBC destruction, or other compared with the reference interval, if there is still hypoxia,
factors that diminish the oxygen-carrying capacity of the blood stimulation of RBC production is warranted because the numbers
(Box 8-3), the production of EPO is increased. present are not meeting the oxygen need. An elevation of RBC
Hypoxia increases EPO production in peritubular cells numbers above the reference interval, erythrocytosis, is seen in
mainly by transcriptional regulation. The EPO gene has a conditions such as lung disease and cardiac disease in which the
hypoxia-sensitive region (enhancer) in its 39 regulatory com- blood is not being well oxygenated. Newborns have higher num-
ponent.15 When oxygen tension in the cell decreases, hypoxia- bers of RBCs because the fetal hemoglobin in their cells does not
inducible factor-1, a transcription factor, is assembled in the unload oxygen to the tissues readily, so newborns are slightly
cytoplasm,16 migrates to the nucleus, and interacts with the 39 hypoxic compared with adults. To compensate, they make more
enhancer of the gene. This results in transcription of more RBCs.
EPO messenger RNA molecules, and production of more EPO. 1. Donnelly S: Why is erythropoietin made in the kidney? The kidney
functions as a “critmeter” to regulate the hematocrit, Adv Exp Med
Erythropoietin Biol 543:73-87, 2003.
Structure.  ​EPO is a thermostable, nondialyzable, glyco- 2. Stewart P: Physiology of the kidney, Update in Anaesthesia 9:24-
protein hormone with a molecular weight of 34 kD.17 It consists 28, 1998. http://www.wfsahq.org/archive-update-in-anaesthesia/
of a carbohydrate unit that reacts specifically with RBC receptors update-in-anaesthesia/update-009/detail. Accessed October 6,
and a terminal sialic acid unit, which is necessary for biological 2014.
activity in vivo.18 On desialation, EPO activity ceases.19
 CHAPTER 8  Erythrocyte Production and Destruction 105

Action.  ​EPO is a true hormone, being produced at one circulating RBCs is by increasing the number of cells that will
location (the kidney) and acting at a distant location (the bone be able to mature into circulating erythrocytes. It does this by
marrow). It is a growth factor (or cytokine) that initiates an decreasing apoptosis, the programmed death of RBC progeni-
intracellular message to the developing RBCs; this process is tors.25,26 To understand this process, an overview of apoptosis
called signal transduction. EPO must bind to its receptor on the in general is helpful.
surface of cells to initiate the signal or message (Figure 33-9). Apoptosis: programmed cell death.  ​As noted previously, it
The receptor is a transmembrane homodimer consisting of two takes about 18 to 21 days to produce an RBC from stimulation
identical polypeptide chains.20 EPO-responsive cells vary in their of the earliest erythroid progenitor (BFU-E) to release from the
sensitivity to EPO.21 Some are able to respond to low levels of bone marrow. In times of increased need for RBCs, such as
EPO,22 whereas others require higher levels. In healthy circum- when there is loss from the circulation during hemorrhage, this
stances when RBC production needs to proceed at a modest but time lag would be a significant problem. One way to prepare
regular rate, the cells requiring only low levels of EPO respond. for such a need would be to maintain a store of mature RBCs
If EPO levels rise secondary to hypoxia, however, a larger in the body for emergencies. RBCs cannot be stored in the
population of EPO-sensitive cells is able to respond. body for this sort of eventuality, however, because they have a
The binding of EPO, the ligand, to its receptor on erythro- limited life span. Therefore, instead of storing mature cells for
cyte progenitors initiates a cascade of intracellular events (“the emergencies, the body produces more CFU-Es than needed at
program”) that ultimately leads to cell division, maturation, all times. When there is a basal or steady-state demand for
and more red blood cells entering the circulation. EPO’s effects RBCs, the extra progenitors are allowed to die. When there is
are mediated by Janus-activated tyrosine kinase 2 (JAK2) signal an increased demand for RBCs, however, the RBC progenitors
transducers that are associated with the cytoplasmic domain of have about an 8- to 10-day head start in the production pro-
the EPO receptor and ultimately affect gene expression in the cess. This process of intentional wastage of cells occurs by
RBC nucleus (Figure 33-9).23 EPO has three major effects: al- apoptosis, and it is part of the cell’s genetic program.
lowing early release of reticulocytes from the bone marrow, Process of apoptosis.  ​Apoptosis is a sequential process char-
preventing apoptotic cell death, and reducing the time needed acterized by, among other things, the degradation of chroma-
for cells to mature in the bone marrow. These processes are tin into fragments of varying size that are multiples of 180 to
described in detail in the following sections. The essence is that 185 base pairs long; protein clustering; and activation of trans-
EPO puts more RBCs into the circulation at a faster rate than glutamase. This is in contrast to necrosis, in which cell injury
occurs without its stimulation. causes swelling and lysing with release of cytoplasmic contents
Early Release of Reticulocytes.  ​EPO promotes early that stimulate an inflammatory response (Chapter 6). Apopto-
release of developing erythroid precursors from the marrow sis is not associated with inflammation.27
by two mechanisms. EPO induces changes in the adventitial During the sequential process of apoptosis, the following
cell layer of the marrow/sinus barrier that increase the width morphologic changes can be seen: condensation of the
of the spaces for RBC egress into the sinus.24 This mechanism nucleus, causing increased basophilic staining of the chroma-
alone, however, is insufficient for cells to leave the marrow. tin; nucleolar disintegration; and shrinkage of cell volume
RBCs are held in the marrow because they express surface with concomitant increase in cell density and compaction of
membrane receptors for adhesive molecules located on the cytoplasmic organelles, while mitochondria remain normal.28
bone marrow stroma. EPO downregulates the expression of This is followed by a partition of cytoplasm and nucleus into
these receptors so that cells can exit the marrow earlier than membrane-bound apoptotic bodies that contain varying
they normally would.8-10 The result is the presence in the cir- amounts of ribosomes, organelles, and nuclear material. The
culation of reticulocytes that are still very basophilic because last stage of degradation produces nuclear DNA fragments
they have not spent as much time degrading their ribosomes consisting of multimers of 180 to 185 base pair segments.
or making hemoglobin as they normally would before enter- Characteristic blebbing of the plasma membrane is observed.
ing the bloodstream. These are called shift reticulocytes be- The apoptotic cell contents remain membrane-bound and are
cause they have been shifted from the bone marrow early ingested by macrophages, which prevents an inflammatory
(Figure 8-8, A). Their bluish cytoplasm with Wright stain is reaction. The membrane-bound vesicles display so-called “eat
evident, so the overall blood picture is said to have polychro- me” signals on the membrane surface (discussed later) that
masia. Even nucleated RBCs (i.e., normoblasts) can be re- promote macrophage ingestion.29
leased early in cases of extreme anemia when the demand for Evasion of apoptosis by erythroid progenitors and precursors. ​
RBCs in the peripheral circulation is great. Releasing cells Thus, under normal circumstances, many red cell progenitors
from the marrow early is a quick fix, so to speak; it is limited will undergo apoptosis. However, when increased numbers of
in effectiveness because the available precursors in the mar- red cells are needed, apoptosis can be avoided. One effect of
row are depleted within several days and still may not be EPO is an indirect avoidance of apoptosis by removing an
enough to meet the need in the peripheral blood for more apoptosis induction signal. Apoptosis of RBCs is a cellular pro-
cells. A more sustained response is required in times of cess that depends on a signal from either the inside or outside
increased need for RBCs in the circulation. of the cell. Among the crucial molecules in the external messag-
Inhibition of Apoptosis.  ​A second, and probably more ing system is the death receptor Fas on the membrane of the
important, mechanism by which EPO increases the number of earliest RBC precursors, while its ligand, FasL, is expressed by
106 PART II  Blood Cell Production, Structure, and Function

more mature RBCs.28,30 When EPO levels are low, cell produc- bluish tinge to the cytoplasm. These cells are true shift reticu-
tion should be at a low rate because hypoxia is not present. The locytes similar to those in Figure 8-8, A, recognizable in the
excess early erythroid precursors should undergo apoptosis. stained peripheral blood film as especially large, bluish cells
This occurs when the older FasL-bearing erythroid precursors, typically lacking central pallor. They also are called stress re-
such as polychromatic normoblasts, cross-link with Fas-marked ticulocytes because they exit the marrow early during condi-
immature erythroid precursors, such as pronormoblasts and tions of bone marrow “stress,” such as in certain anemias.
basophilic normoblasts, which are then stimulated to undergo EPO also can reduce the time it takes for cells to mature in
apoptosis.28 As long as the more mature cells with FasL are the marrow by reducing individual cell cycle time, specifically
present in the marrow, erythropoiesis is subdued. If the FasL- the length of time that cells spend between mitoses.39 This
bearing cells are depleted, as when EPO stimulates early mar- effect is only about a 20% reduction, however, so that the nor-
row release, the younger Fas-positive precursors are allowed to mal transit time in the marrow of approximately 6 days from
develop, which increases the overall output of RBCs from the pronormoblast to erythrocyte can be shortened by only about
marrow. Thus early release of older cells in response to EPO 1 day by this effect.
indirectly allows more of the younger cells to mature. With the decreased cell cycle time and fewer mitotic divi-
A second mechanism for escaping apoptosis exists for sions, the time it takes from pronormoblast to reticulocyte can
RBC progenitors: direct EPO rescue from apoptosis. This is be shortened by about 2 days total. If the reticulocyte leaves the
the major way in which EPO is able to increase RBC produc- marrow early, another day can be saved, and the typical 6-day
tion. When EPO binds to its receptor on the CFU-E, one of transit time is reduced to fewer than 4 days under the influence
the effects is to reduce production of Fas ligand.31 Thus the of increased EPO.
younger cells avoid the apoptotic signal from the older cells.
Additionally, EPO is able to stimulate production of various Measurement of Erythropoietin.  ​Quantitative mea-
anti-apoptotic molecules, which allows the cell to survive surements of EPO are performed on plasma and other body
and mature.31,32 The cell that has the most EPO receptors and fluids. EPO can be measured by chemiluminescence. Although
is most sensitive to EPO rescue is the CFU-E, although the the reference interval for each laboratory varies, an example
late BFU-E and early pronormoblast have some receptors.33 reference interval is 4 to 27 mU/L.40 Increased amounts of EPO
Without EPO, the CFU-E does not survive.34 in the urine are expected in most patients with anemia, with
The binding of EPO to its transmembrane receptors on the exception of patients with anemia caused by renal disease.
erythroid progenitors and precursors activates JAK2 protein
associated with its cytoplasmic domain (Figure 33-9). Acti- Therapeutic Uses of Erythropoietin.  Recombinant
vated JAK2 then phosphorylates (activates) the signal trans- erythropoietin is used as therapy in certain anemias such as
duction and activator of transcription (STAT) pathway, leading those associated with chronic kidney disease and chemo-
to the production of the anti-apoptotic molecule Bcl-XL (now therapy. It is also used to stimulate RBC production prior to
called Bcl-2 like protein 1).31,32 EPO-stimulated cells develop autologous blood donation and after bone marrow trans-
this molecule on their mitochondrial membranes, preventing plantation. The indications for EPO therapy are summarized
release of cytochrome c, an apoptosis initiator.35 EPO’s effect in Table 7-2.
is mediated by the transcription factor GATA-1, which is Unfortunately, some athletes illicitly use EPO injections to
essential to red cell survival.36 increase the oxygen-carrying capacity of their blood to enhance
Reduced marrow transit time.  ​Apoptosis rescue is the endurance and stamina, especially in long-distance running
major way in which EPO increases RBC mass—by increasing and cycling. The use of EPO is one of the methods of blood
the number of erythroid cells that survive and mature to enter doping, and aside from being banned in organized sports events,
the circulation. Another effect of EPO is to increase the rate it increases the RBC count and blood viscosity to dangerously
at which the surviving precursors can enter the circulation. This high levels and can lead to fatal arterial and venous thrombosis.
is accomplished by two means: increased rate of cellular
processes and decreased cell cycle times. Other Stimuli to Erythropoiesis
EPO stimulates the synthesis of RBC RNA and effectively In addition to tissue hypoxia, other factors influence RBC
increases the rate of the developmental “program.” Among production to a modest extent. It is well documented that
the processes that are accelerated is hemoglobin produc- testosterone directly stimulates erythropoiesis, which par-
tion.37 As mentioned earlier, another accelerated process is tially explains the higher hemoglobin concentration in men
bone marrow egress with the loss of adhesive receptors and than in women.41 Also, pituitary42 and thyroid43 hormones
the acquisition of egress-promoting surface molecules.38 The have been shown to affect the production of EPO and so have
other process that is accelerated is the cessation of division. indirect effects on erythropoiesis.
Cell division takes time and would delay entry of cells to the
circulation, so cells enter cell cycle arrest sooner. As a result,
MICROENVIRONMENT OF THE BONE
the cells spend less time maturing in the marrow. In the cir-
MARROW
culation, such cells are larger due to lost mitotic divisions,
and they do not have time before entering the circulation to The microenvironment of the bone marrow is described in
dismantle the protein production machinery that gives the Chapter 7, and the cytokines essential to hematopoiesis are
 CHAPTER 8  Erythrocyte Production and Destruction 107

discussed there. Here, the details pertinent to erythropoiesis

(i.e., the erythropoietic inductive microenvironment) are
emphasized, including the locale and arrangement of ery-
throid cells and the anchoring molecules involved.
Hematopoiesis occurs in marrow cords, essentially a loose
arrangement of cells outside a dilated sinus area between the
arterioles that feed the bone and the central vein that returns
blood to efferent veins. Erythropoiesis typically occurs in what
are called erythroid islands (Figure 7-6). These are macrophages
surrounded by erythroid precursors in various stages of devel-
opment. 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.
However, since developing RBCs obtain iron via transferrin
(Chapter 11), no direct contact with macrophages is needed for
this. Macrophages are now known to elaborate cytokines that
are vital to the maturation process of the RBCs.44-46 RBC
precursors would not survive without macrophage support via
such stimulation.
A second role for macrophages in erythropoiesis also has Figure 8-11  ​Egress of a red blood cell through a pore in an endothelial
been identified. Although movement of cells through the cell of the bone marrow venous sinus. Arrowheads indicate the endothelial
marrow cords is sluggish, developing cells would exit the cell junctions. (From DeBruyn PPH: Structural substrates of bone marrow
marrow prematurely in the outflow were it not for an anchor- function, Semin Hematol 18:182, 1981.)
ing system within the marrow that holds them there until
development is complete. There are three components to the senescence, which culminates in phagocytosis by macrophages.
anchoring system: a stable matrix of accessory and stromal This is the major way in which RBCs die normally.
cells to which normoblasts can attach, bridging (adhesive)
molecules for that attachment, and receptors on the erythro- Macrophage-Mediated Hemolysis
cyte membrane. (Extravascular Hemolysis)
The major cellular anchor for the RBCs is the macrophage. At any given time, a substantial volume of blood is in the
Several systems of adhesive molecules and RBC receptors tie spleen, which generates an environment that is inherently
the developing RBCs to the macrophages.44 At the same time, stressful on cells. Movement through the red pulp is sluggish.
RBCs are anchored to the extracellular matrix of the bone The available glucose in the surrounding plasma is depleted
marrow, chiefly by fibronectin.9 quickly as the cell flow stagnates, so glycolysis slows. The pH is
When it comes time for the RBCs to leave the marrow, low, which promotes iron oxidation. Maintaining reduced iron
they cease production of the receptors for the adhesive is an energy-dependent process, so factors that promote iron
molecules.9 Without the receptor, the cells are free to move oxidation cause the RBC to expend more energy and accelerate
from the marrow into the venous sinus. Entering the venous the catabolism of enzymes.
sinus requires the RBC to traverse the barrier created by the In this hostile environment, aged RBCs succumb to the
adventitial cells on the cord side, the basement membrane, various stresses. Their deteriorating glycolytic processes lead
and the endothelial cells lining the sinus. Egress through this to reduced ATP production, which is complicated further by
barrier occurs between adventitial cells, through holes (fen- diminished amounts of available glucose. The membrane
estrations) in the basement membrane, and through pores in systems that rely on ATP begin to fail. Among these are
the endothelial cells19 (Figure 8-11).24,47,48 enzymes that maintain the location and reduction of phos-
pholipids of the membrane. Lack of ATP leads to oxidation of
membrane lipids and proteins. Other ATP-dependent
ERYTHROCYTE DESTRUCTION
enzymes are responsible for maintaining the high level of
All cells experience the deterioration of their enzymes over intracellular potassium while pumping sodium out of the
time due to natural catabolism. Most cells are able to replenish cells. As this system fails, intracellular sodium increases and
needed enzymes and continue their cellular processes. As a potassium decreases. The effect is that the selective permea-
nonnucleated cell, however, the mature erythrocyte is unable bility of the membrane is lost and water enters the cell. The
to generate new proteins, such as enzymes, so as its cellular discoid shape is lost and the cell becomes a sphere.
functions decline, the cell ultimately approaches death. The RBCs must remain highly flexible to exit the spleen by
average RBC has sufficient enzyme function to live 120 days. squeezing through the so-called splenic sieve formed by the
Because RBCs lack mitochondria, they rely on glycolysis for endothelial cells lining the venous sinuses and the basement
production of adenosine triphosphate (ATP). The loss of glyco- membrane. Spherical RBCs are rigid and are not able to
lytic enzymes is central to this process of cellular aging, called squeeze through the narrow spaces; they become trapped
108 PART II  Blood Cell Production, Structure, and Function

cells and distinguish them from younger cells; thus the older
cells are targeted for ingestion and lysis.
When an RBC lyses within a macrophage, the major com-
ponents are catabolized. The iron is removed from the heme.
It can be stored in the macrophage as ferritin until transported
out. The globin of hemoglobin is degraded and returned to the
metabolic amino acid pool. The protoporphyrin component of
heme is degraded through several intermediaries to bilirubin,
which is released into the plasma and ultimately excreted by
the liver in bile. The details of bilirubin metabolism are dis-
Figure 8-12  ​Macrophage ingesting a spherocytic erythrocyte. (From cussed in Chapter 23.
Bessis M: Corpuscles, atlas of RBC shapes, New York, 1974, Springer-
Verlag.) Mechanical Hemolysis (Fragmentation or
Intravascular Hemolysis)
against the endothelial cells and basement membrane. In this Although most natural RBC deaths occur in the spleen, a small
situation, they are readily ingested by macrophages that patrol portion of RBCs rupture intravascularly (within the lumen of
along the sinusoidal lining (Figure 8-12). blood vessels). The vascular system can be traumatic to RBCs,
Some researchers view erythrocyte death as a nonnucleated with turbulence occurring in the chambers of the heart or at
cell version of apoptosis, termed eryptosis,49 that is precipitated points of bifurcation of vessels. Small breaks in blood vessels and
by oxidative stress, energy depletion, and other mechanisms that resulting clots can also trap and rupture cells. The intravascular
create membrane signals that stimulate phagocytosis. It is highly rupture of RBCs from purely mechanical or traumatic stress re-
likely that there is no single signal but rather that macrophages sults in fragmentation and release of the cell contents into the
recognize several. Examples of the signals generating continuing plasma; this is called fragmentation or intravascular hemolysis.
research interest include binding of autologous immunoglobu- When the membrane of the RBC has been breached, regardless
lin G (IgG) to band-3 membrane protein clusters, exposure of of where the cell is located when it happens, the cell contents
phosphatidylserine on the exterior (plasma side) of the mem- enter the surrounding plasma. Although mechanical lysis is a rela-
brane, and inability to maintain cation balance.50 Senescent tively small contributor to RBC demise under normal circum-
changes to leukocyte surface antigen CD47 (integrin-associated stances, the body still has a system of plasma proteins, including
protein) may also be involved by binding thrombospondin-1, haptoglobin and hemopexin, to salvage the released hemoglobin
which then provides an “eat me” signal to macrophages.51 What- so that its iron is not lost in the urine. Hemolysis and the func-
ever the signal, macrophages are able to recognize senescent tions of haptoglobin and hemopexin are discussed in Chapter 23.

SUMMARY

• RBCs develop from committed erythroid progenitor cells in the • EPO, the primary hormone that stimulates the production of eryth-
bone marrow, the BFU-E and CFU-E. rocytes, is able to rescue the CFU-E from apoptosis, shorten the
• The morphologically identifiable precursors of mature RBCs, in time between mitoses of precursors, release reticulocytes from the
order from youngest to oldest, are the pronormoblast, basophilic marrow early, and reduce the number of mitoses of precursors.
normoblast, polychromatic normoblast, orthochromic normoblast, • Apoptosis is the mechanism by which an appropriate normal
and polychromatic erythrocyte or reticulocyte. production level of cells is controlled. Fas, the death receptor, is
• As erythroid precursors age, the nucleus becomes condensed and expressed by young normoblasts, and FasL, the ligand, is ex-
ultimately is ejected from the cell, which produces the polychro- pressed by older normoblasts. As long as older cells mature slowly
matic erythrocyte or reticulocyte stage. The cytoplasm changes in the marrow, they induce the death of unneeded younger cells.
color from blue, reflecting numerous ribosomes, to salmon-pink as • EPO rescues cells from apoptosis by stimulating the production of
hemoglobin accumulates and the ribosomes are degraded. Each anti-apoptotic molecules that counteract the effects of Fas and
stage can be identified by the extent of these nuclear and cyto- FasL and simultaneously decreasing Fas production by young
plasmic changes. normoblasts.
• It takes approximately 18 to 21 days for the BFU-E to mature to an • Survival of RBC precursors in the bone marrow depends on adhe-
RBC, of which about 6 days are spent as identifiable precursors in sive molecules, such as fibronectin, and cytokines that are elabo-
the bone marrow. The mature erythrocyte has a life span of rated by macrophages and other bone marrow stromal cells. RBCs
120 days in the circulation. are found in erythroid islands, where erythroblasts at various
• Hypoxia of peripheral blood is detected by the peritubular fibro- stages of maturation surround a macrophage.
blasts of the kidney, which upregulates transcription of the EPO • As RBC precursors mature, they lose adhesive molecule receptors
gene to increase the production of EPO. and can leave the bone marrow. Egress occurs between
 CHAPTER 8  Erythrocyte Production and Destruction 109

adventitial cells but through pores in the endothelial cells of the of phosphatidylserine on the outer membrane, cation balance
venous sinus. changes, and CD47-thrombospondin 1 binding.
• Aged RBCs, or senescent cells, cannot regenerate catabolized • Fragmentation or intravascular hemolysis results when mechani-
enzymes because they lack a nucleus. The semipermeable mem- cal factors rupture the cell membrane while the cell is in the pe-
brane becomes more permeable to water, so the cell swells and ripheral circulation. This pathway accounts for a minor component
becomes spherocytic and rigid. It becomes trapped in the splenic of normal destruction of RBCs.
sieve.
• Extravascular or macrophage-mediated hemolysis accounts for Now that you have completed this chapter, go back and
most normal RBC death. The signals to macrophages that initiate read again the case study at the beginning and respond
RBC ingestion may include binding of autologous IgG, expression to the questions presented.

R E V I E W Q UESTIONS
Answers can be found in the Appendix. 6. In the bone marrow, RBC precursors are located:
a. In the center of the hematopoietic cords
1. Which of the following is an erythrocyte progenitor? b. Adjacent to megakaryocytes along the adventitial cell
a. Pronormoblast lining
b. Reticulocyte c. Surrounding fat cells in apoptotic islands
c. CFU-E d. Surrounding macrophages in erythroid islands
d. Orthochromic normoblast
7. Which of the following determines the timing of egress of
2. Which of the following is the most mature normoblast? RBCs from the bone marrow?
a. Orthochromic normoblast a. Maturing normoblasts slowly lose receptors for adhe-
b. Basophilic normoblast sive molecules that bind them to stromal cells.
c. Pronormoblast b. Stromal cells decrease production of adhesive mole-
d. Polychromatic normoblast cules over time as RBCs mature.
c. Endothelial cells of the venous sinus form pores at
3. What erythroid precursor can be described as follows: the specified intervals of time, allowing egress of free cells.
cell is of medium size compared with other normoblasts, d. Periodic apoptosis of pronormoblasts in the marrow
with an N:C ratio of nearly 1:1. The nuclear chromatin is cords occurs.
condensed and chunky throughout the nucleus. No nu-
cleoli are seen. The cytoplasm is a muddy, blue-pink 8. What single feature of normal RBCs is most responsible for
color. limiting their life span?
a. Reticulocyte a. Loss of mitochondria
b. Pronormoblast b. Increased flexibility of the cell membrane
c. Orthochromic normoblast c. Reduction of hemoglobin iron
d. Polychromatic normoblast d. Loss of the nucleus

4. Which of the following is not related to the effects of eryth- 9. Intravascular or fragmentation hemolysis is the result of
ropoietin? trauma to RBCs while in the circulation.
a. The number of divisions of a normoblast a. True
b. The formation of pores in sinusoidal endothelial cells b. False
for marrow egress
c. The time between mitoses of normoblasts 10. Extravascular hemolysis occurs when:
d. The production of antiapoptotic molecules by erythroid a. RBCs are mechanically ruptured
progenitors b. RBCs extravasate from the blood vessels into the tissues
c. Splenic macrophages ingest senescent cells
5. Hypoxia stimulates RBC production by: d. Erythrocytes are trapped in blood clots outside the
a. Inducing more pluripotent stem cells into the erythroid blood vessels
lineage
b. Stimulating EPO production by the kidney
c. Increasing the number of RBC mitoses
d. Stimulating the production of fibronectin by macro-
phages of the bone marrow
110 PART II  Blood Cell Production, Structure, and Function

11. A pronormoblast in its usual location belongs to the RBC 12. A cell has an N:C ratio of 4:1. Which of the following state-
mass of the body, but not to the erythron. ments would describe it?
a. True a. The bulk of the cell is composed of cytoplasm.
b. False b. The bulk of the cell is composed of nucleus.
c. The proportions of cytoplasm and nucleus are roughly
equal.

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& Anastasi, J. (Eds.), Hematology: Basic Principles and Practice. 20. Schneider, H., Chaovapong, W., Matthews, D. J., et al.
(6th ed., pp. 258–279). Philadelphia: Elsevier/Saunders. (1997). Homodimerization of erythropoietin receptor by
2. Shumacher, H. R., & Erslev, A. J. (1956). Bone marrow kinetics. a bivalent monoclonal anibody triggers cell proliferation
In Szirmani, E., (Ed.), Nuclear Hematology. (pp. 89–132). New and differentiation of erythroid precursors. Blood, 89(2),
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