MLS 113A LESSON | 02

HEMATOLOGY Lecture | AJDC | Batch 2024

MATURATION PROCESS
TERMINOLOGY
● Erythrocytes - RBCs
● Erythroblasts – the nucleated precursors in the bone marrow. They also

may be called normoblasts, which refers to developing nucleated cells

(i.e., blasts) with normal appearance.
○This is in contrast to the abnormal appearance of the developing
nucleated cells in megaloblastic anemia, in which the erythroblasts are
called megaloblasts because of their large size.
○Megaloblasts - big RBCs; not immature (found in the circulation when
there is a nutritional deficiency); folate or vitamin B12 deficiency

Three nomenclatures are used for naming the erythroid precursors:

Erythroblast
used primarily in Europe.
terminology

used more often in the United States, it has the

Normoblastic
advantage of being descriptive of the
terminology
appearance of the cells.
ERYTHROID PROGENITORS
Rubriblast parallels the nomenclature used for granulocyte Burst-forming unit-erythroid (BFU-E) and Colony-forming unit-
terminology development (granulopoiesis) erythroid (CFU-E)
● The morphologically identifiable erythrocyte precursors develop from two

functionally identifiable progenitors, both committed to the erythroid cell

RUBRIBLAST NORMOBLAST ERYTHROBLAST line.
Rubriblast Pronormoblast Proerythroblast ● It takes about one week for the BFU-E to mature to the CFU-E and another

Basophilic week for the CFU-E to become a pronormoblast, which is the first
Basophilic Normoblast/ Erythroblast/ morphologically identifiable RBC precursor.
Prorubricyte
EARLY NORMOBLAST EARLY ● While at the CFU-E stage, the cell completes approximately three to five
ERYTHROBLAST divisions before maturing further.
Polychromatic Polychromatic ● It takes approximately another 6 to 7 days for the precursors to become

Rubricyte Normoblast/ Erythroblast/ mature enough to enter the circulation, so approximately 18 to 21 days
INTERMEDIATE
INTERMEDIATE NORMOBLAST ERYTHROBLAST are required to produce a mature RBC from the BFU-E.
Orthochromatic
Orthochromatic Normoblast/ Erythroblast/ 7 days 7 days 6-7 days
Metarubricyte BFU-E → CFU-E → pronormoblast → mature RBC
LATE NORMOBLAST LATE
ERYTHROBLAST
● Reticulocyte (Supravital Stain) ERYTHROID PRECURSORS
● Polychromatophilic Erythrocyte (Wright-stained Smear) Normoblastic proliferation, similar to the proliferation of other cell lines, is
● Diffusely Basophilic Erythrocyte (Wright-stained Smear) a process encompassing replication (i.e., division) to increase cell numbers
and development from immature to mature cell stages.

Pronormoblast
● The earliest morphologically recognizable erythrocyte precursor. is

derived via the BFU-E and CFU-E from the pluripotential stem cells.
● 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 polychromatic normoblast.

In the erythrocyte cell line, there are typically three and occasionally as
many as five divisions with subsequent nuclear and cytoplasmic maturation
of the daughter cells, so from a single pronormoblast, 8 to 32 mature RBCs
usually result (23 or 25)
 MLS 113A LESSON | 02
HEMATOLOGY Lecture | AJDC | Batch 2024
white appearance of the parachromatin. This chromatin/parachromatin
CRITERIA USED IN THE IDENTIFICATION OF THE distinction is more dramatic than in other cell lines. Ultimately, the
ERYTHROID PRECURSORS nucleus becomes quite condensed, with no parachromatin evident at all,
• Morphologic identification of blood cells depends on a well-stained and the nucleus is said to be pyknotic.
peripheral blood film or bone marrow smear. Romanowsky stain, such
as Wright or Wright- Giemsa, is commonly used. 4. Nucleoli disappear. Nucleoli represent areas where the ribosomes are
• careful examination of the nucleus and the cytoplasm. The qualities of formed and are seen early in cell development as cells begin actively
greatest importance in the identification of RBCs are the: synthesizing proteins. As RBCs mature, the nucleoli disappear, which
precedes the ultimate cessation of protein synthesis.
1. nuclear chromatin pattern (texture, density, homogeneity)
2. nuclear diameter, nucleus: cytoplasm (N:C) ratio
-(N:C - nucleus to cytoplasmic ratio)
3. presence or absence of nucleoli
-younger cells, present nucleoli;
-older, no nucleolus anymore
4. cytoplasmic color (blue to red)

5. The cytoplasm changes from blue to gray-blue to salmon pink.

• chromatin clumped, homogeneous, white portions BLUENESS OR BASOPHILIA

• rRNA - prominent nucleus • due to acidic components that attract the basic stain, such as methylene
• reticulocytes; extreme anemia blue.
• The degree of cytoplasmic basophilia correlates with the amount of
ribosomal RNA. These organelles decline over the life of the developing
NUCLEUS-TO-CYTOPLASM (N:C) RATIO RBC, and the blueness fades.
The ratio is a visual estimate of what area of the cell is occupied by the
nucleus compared with the cytoplasm. Methylene blue basic; attracts acid
Eosin acidic; attracts basic
If the areas of each are
N:C ratio is 1:1
approximately equal

the proportion of the nucleus is

If the nucleus takes up less
lower, and the ratio is lower
than 50% of the area of the cell,
(e.g., 1:5 or less than 1).

If the nucleus takes up more

the ratio is higher (e.g., 3:1 or 3)
than 50% of the area of the cell

In the red blood cell line, the proportion of the nucleus shrinks as the cell
matures and the cytoplasm increases proportionately, although the overall EOSINOPHILIA OR ACIDOPHILIA
cell diameter grows smaller. In short, the N:C ratio decrease ● Pinkness is due to the accumulation of more basic components that attract

the acid stain eosin.

As RBCs mature, several general trends affect their appearance and
graphically represents these trends.,
1. The overall diameter of the cell decreases.
2. The diameter of the nucleus decreases more rapidly than does the
size of the cell, N:C ratio also decreases.
3. The nuclear chromatin pattern becomes coarser, clumped, and
condensed.
● correlates with the accumulation of hemoglobin as the cell matures.
● the cell starts out being active in protein production on the ribosomes that
make the cytoplasm basophilic, transitions through a period in which the
red of hemoglobin begins to mix with that blue, and ultimately ends with a
thoroughly salmon pink color when the ribosomes are gone and only
hemoglobin remains.

The nuclear chromatin of

RBCs is inherently coarser
than that of myeloid
precursors. 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
 MLS 113A LESSON | 02
HEMATOLOGY Lecture | AJDC | Batch 2024
MATURATION SEQUENCE
1. PRONORMOBLAST 3. POLYCHROMATIC (POLYCHROMATOPHILIC) NORMOBLAST
NUCLEUS NUCLEUS
• N:C ratio of 8:1 • chromatin pattern varies during this stage of

• round to oval, containing one or two nucleoli. development, showing some openness early in
• purple red chromatin is open and contains few, if any, fine clumps. the stage but becoming condensed by the end.
• The condensation of chromatin reduces the

CYTOPLASM diameter of the nucleus considerably, so the

• dark blue because of the concentration of N:C ratio decreases from 4:1 to about 1:1 by the
ribosomes end of the stage.
• golgi complex may be visible next to the • no nucleoli are present.

nucleus as a pale, unstained area.

• may show small tufts of irregular cytoplasm CYTOPLASM
along the periphery of the membrane. • first stage in which the pink color associated with stained hemoglobin can

be seen. The stained color reflects the accumulation of hemoglobin

DIVISION pigmentation over time and concurrent decreasing amounts of RNA.
• undergoes mitosis and gives rise to two • The color produced is a mixture of pink and blue, resulting in a murky gray-

daughter pronormoblasts. blue. The stage’s name refers to this combination of multiple colors,
because polychromatophilic means “many color loving.”
LOCATION
• present only in the bone marrow in healthy DIVISION
states. • the last stage in which the cell is capable of

undergoing mitosis, although likely only early in

CELLULAR ACTIVITY the stage.
• begins to accumulate the components necessary for hemoglobin • goes through mitosis, producing daughter cells

production. that mature and develop into orthochromic

• proteins and enzymes necessary for iron uptake and protoporphyrin normoblasts.
synthesis are produced.
• globin production begins. LOCATION
• present only in the bone marrow in healthy states.

LENGTH OF TIME IN THIS STAGE

• more than 24 hours. CELLULAR ACTIVITY
• Hemoglobin synthesis increases, and the accumulation begins to be

visible in the color of the cytoplasm.

2. BASOPHILIC NORMOBLAST • Cellular organelles are still present, particularly ribosomes, which
NUCLEUS contribute a blue aspect to the cytoplasm. The progressive condensation
• The chromatin begins to condense, of the nucleus and disappearance of nucleoli are evidence of progressive
revealing clumps along the periphery of the decline in transcription of deoxyribonucleic acid (DNA).
nuclear membrane and a few in the interior.
• the parachromatin areas become larger and
LENGTH OF TIME IN THIS STAGE
sharper • This stage lasts approximately 30 hours.
• N:C ratio decreases to about 6:1.

• chromatin stains deep purple-red. Nucleoli

may be present early in the stage but 4. ORTHOCHROMIC NORMOBLAST

disappear later. NUCLEUS
• The nucleus is completely condensed (i.e.,
CYTOPLASM pyknotic) or nearly so. As a result, the N:C ratio
• the cytoplasm may be a deeper, richer blue than in the pronormoblast—
is low or approximately 1:2.
hence the name basophilic for this stage.

DIVISION CYTOPLASM
• undergoes mitosis, giving rise to two daughter cells.
• The increase in the salmon-pink color of the cytoplasm reflects nearly

complete hemoglobin production. The residual ribosomes react with the

LOCATION basic component of the stain and contribute a slightly bluish hue to the
• is present only in the bone marrow in healthy states.
cell, but that fades toward the end of the stage as the organelles are
degraded.
CELLULAR ACTIVITY
• detectable hemoglobin synthesis occurs, but
DIVISION
the many cytoplasmic organelles, including • The orthochromic normoblast is not capable of division due to the
ribosomes and a substantial amount of condensation of the chromatin.
messenger ribonucleic acid (RNA; chiefly for
hemoglobin production), completely mask LOCATION
the minute amount of hemoglobin • The orthochromic normoblast is present only in the bone marrow in healthy
pigmentation. states.
LENGTH OF TIME IN THIS STAGE
• slightly more than 24 hours.
 MLS 113A LESSON | 02
HEMATOLOGY Lecture | AJDC | Batch 2024

CELLULAR ACTIVITY DIVISION

• Lacking a nucleus, the polychromatic erythrocyte cannot divide.

LOCATION
• The polychromatic erythrocyte resides in the bone marrow for 1 day or

longer and then moves into the peripheral blood for about 1 day before
reaching maturity.
• During the first several days after exiting the marrow, the polychromatic

erythrocyte is retained in the spleen for pitting of inclusions and membrane

• Hemoglobin production continues on the remaining ribosomes. polishing by splenic macrophages, which results in the biconcave discoid
• Late in this stage, the nucleus is ejected from the cell. mature RBC.
• The nucleus moves to the cell membrane and into a pseudopod-like

projection. As part of the maturation program, loss of vimentin, is probably CELLULAR ACTIVITY
important in the movement of the nucleus to the cell periphery. • completes production of
hemoglobin from residual
• Vimentin- a protein responsible for holding organelles in proper location messenger RNA using the
in the cytoplasm remaining ribosomes.
• cytoplasmic protein production
• Ultimately, the nucleus-containing projection separates from the cell by machinery is simultaneously being
having the membrane seal and pinch off the projection with the nucleus dismantled.
enveloped by cell membrane. • Endoribonuclease digests the
• Nonmuscle myosin of the membrane is important in this pinching process. ribosomes.
• The enveloped extruded nucleus, called a pyrenocyte, is then engulfed • acidic components that attract the basophilic stain decline during this

by bone marrow macrophages. The macrophages recognize stage to the point that the polychromatophilia is not readily evident in the
phosphatidlyserine on the pyrenocyte surface as an “eat me” flag. polychromatic erythrocytes on a normal peripheral blood film 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 film is made.
• The residual ribosomes appear as a mesh of small blue strands, a

reticulum, or, when more fully digested, merely blue dots. When so
stained, the polychromatic erythrocyte is called a reticulocyte.

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. Based on the Wright-stained appearance, the reticulocyte is
called a polychromatic erythrocyte because it lacks a nucleus and is no
• Other organelles are extruded and longer an erythroblast but has a bluish tinge.
ingested in similar fashion. Often, • When polychromatic erythrocytes are prominent on a peripheral blood film,

small fragments of nucleus are left the examiner uses the comment polychromasia or polychromatophilia.
behind if the projection is pinched off • A second functional change in polychromatic erythrocytes is the reduced

before the entire nucleus is production of receptors for the adhesive molecules that hold developing
enveloped. These fragments are RBCs in the marrow. As these receptors decline, cells are freed to leave
called Howell-Jolly bodies when seen the marrow.
in peripheral blood cells and are
typically removed from the cells by LENGTH OF TIME IN THIS STAGE
the splenic macrophage pitting • The cell typically remains a polychromatic erythrocyte for about 3 days,

process once the cell enters the with the first 2 days spent in the marrow and the third spent in the
circulation. peripheral blood, although possibly sequestered in the spleen.

LENGTH OF TIME IN THIS STAGE

• This stage lasts approximately 48 hours. 7. ERYTHROCYTE
NUCLEUS.
• No nucleus is present in mature RBCs.

6. POLYCHROMATIC (POLYCHROMATOPHILIC)
ERYTHROCYTE OR RETICULOCYTE CYTOPLASM.
NUCLEUS • The mature circulating erythrocyte is a
• no nucleus. biconcave disc measuring 7 to 8 μm in diameter,
with a thickness of about 1.5 to 2.5 μm.
CYTOPLASM. • On a stained blood film, it appears as a salmon

• predominant color is that of hemoglobin. pink-staining cell with a central pale area that
• By the end of the polychromatic erythrocyte corresponds to the concavity.
stage, the cell is the same color as a mature • The central pallor is about one third the dia meter

RBC, salmon pink. It remains larger than a of the cell.

mature cell, however. shape of the cell is not
the mature biconcave disc but is irregular in electron micrographs.
 MLS 113A LESSON | 02
HEMATOLOGY Lecture | AJDC | Batch 2024

DIVISION.
• The erythrocyte cannot divide.

LOCATION AND LENGTH OF TIME IN THIS STAGE.

• Mature RBCs remain active in the circulation for approximately 120 days.

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 interior of the erythrocyte contains

mostly hemoglobin, the oxygen-carrying component. 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.
• 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.
• RBCs must squeeze through small spaces such as the basement

membrane of the bone marrow venous sinus., 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.