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.