Dr.

Talaat Mirza Myelopoiesis 10 pages

MYELOPOIESIS

Myelopoiesis is the process of making new WHITE blood cells (myeloid cells).

There are two major types of myeloid cells: 1- granuloid, and 2- non-granuloid. As

the names imply, granuloid cells have granules (in the cytoplasm) where as non-

granuloid are devoid of such granules.

GRANULOID CELLS

Three sub types of granuloid cells are present in BM and peripheral blood (pb):

1- The Poly-Morphonuclear-Neutrophil (pmn) and the band (the stage preceding

the pmn in maturation) compromising up to 70% of pb. These cells have very

fine (minute) granules all over the cytoplasm. The color of these granules is

identical to the color of the RBC's.

2- The basophil making up to 1% of pb. These cells are characterized by the

presence of very large dark blue-black granules all over the cell. The

granules are very basophilic hence the color is very dark blue.

3- The eosinophil making up 1-5% of pb. These cells are characterized by the

presence of very large bright red or pink granules all over the cytoplasm.

The granules are very acidophilic, hence the color is very bright red acquired

by taking up the eosin dye.

NON-GRANULOID CELLS

Two major groups of cells are non-granuloid: 1- the lymphoid cells, and 2- the

monocytoid/macrophage ones. In this section of the myelopoiesis, only

monocytes/macrophages development and differentiation is discussed. Lymphoid

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Dr. Talaat Mirza Myelopoiesis 10 pages

cells will not be discussed due to their different stem cell origin. Lymphpoiesis is

going to be discussed in detail, in the next section.

COMMITMENT TO DIFFERENTIATION

The pluripotent stem cell differentiates into a more specialized cell known as the

CFU-GEMM. The CFU-GEMM (also known as CFU-mix) is the earliest form of

stem cell committed to myeloid differentiation. As mentioned in the previous lecture,

CFU-GEMM can give rise to only 4 cell lines (granuloid, erythroid, monocytoid, and

megakaryocytoid). Specific myeloid growth factors such as G-CSF, M-CSF, and

GM-CSF act on the CFU-GEMM to enhance its differentiation to one of these

myeloid cell lines. On the other hand, CFU-GEMM's exposed to erythropoietin

(especially in the absence of G-CSF, M-CSF, or GM-CSF) would differentiate to

erythroid cells. Erythroid differentiation is going to be discussed in detail, in later

lectures.

Once the CFU-GEMM is exposed to G-CSF and other supporting GF’s, the stem cell

further commits it self to granuloid differentiation ending in neutrophils, basophils,

and/or eosinophils. Similarly, if it was exposed to M-CSF and other supporting

GF’s, the CFU-GEMM further commits it self to monocyte/macrophage

differentiation ending in monocytes and/or macrophages.

Important features in morphological evaluation

The following considerations are to be followed whenever a cell is to be evaluated or

identified, morphologically:

1- Cell size: normal red blood cells have a diameter of about 7 microns (μ). One

should use the RBC as a reference of size. For example, a small resting

lymphocyte is about 9 microns (therefore slightly bigger than a normal RBC)

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however a mature monocyte is about 17 microns (more than twice the size of a

normal RBC).

2- Shape: the shape is evaluated noting criteria such as, shape of nucleus (round,

oval, or irregular), color (of nucleus & cytoplasm), regularity of the cytoplasm,

multi lobulated nucleus versus single, etc.

3- Nucleus: Size and shape of the nucleus must be evaluated noting whether it is

single or multiple. Also, the appearance of chromatin (smooth or condensed).

The more condensed the chromatin, the more mature the cell (immature

cells, e.g., blasts, have very smooth chromatin). Presence or absence of

nucleoli should be observed because mature cells do not have nucleoli.

4- Cytoplasm: color (light blue or gray, dark blue, reddish, etc.), presence or

absence of granules, presence of vacuoles, shape of cytoplasm (regular versus

irregular), presence of appendages (projections) from the cytoplasm, presence

of strange looking bodies (inclusions), etc.

5- Nucleus to Cytoplasm (N/C, also N:C) ratio: early immature cells are

characterized by extremely large nuclei, that is, their nucleus could occupy

up to 95% off cell size with very little cytoplasm evident. As cells mature,

nucleus size decreases and cytoplasm increases to reach about 50% or even

less in certain cells, e.g., plasma cell (a type of B-lymphocyte, to be discussed

in detail later).

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MATURATION SEQUENCE

The first step in myeloid differentiation is the commitment of the pluripotent stem to

become the multipotent stem cell (CFU-GEMM, also known as CFU-mix). The

CFU-GEMM then differentiates furthermore to give rise to the first morphologically

identified myeloid cell, the Myeloblast. From the myeloblast, five morphologically

different maturation forms are produced (not including the blast itself). Therefore, the

stages in the myeloid differentiation are: 1- myeloblast, 2- promyelocyte, 3-

myelocyte, 4- metamyelocyte, 5- band, and 6- neutrophil (pmn). These sequences

also exist for the basophil and the eosinophil, however, in writing them, the words

basophilic or eosinophilic precede the maturation stage, e.g., basophilic promyelocyte,

or eosinophilic metamyelocyte, etc. Morphologically, blasts are only identified as

BLASTS without any specification of the type whether, granuloid, monocytoid,

lymphoid, or else (see below further explanation).

1- The myeloblast: This is the earliest morphologically identified myeloid cell.

The blast is impossible to distinguish from a CFU-GEMM or even a CFU-

S (the pluripotent) stem cell on the basis of morphology only. Only CD

markers, molecular, and biological assays can differentiate among these cells.

It is a very large cell of up to 20 μ in size (about 3 times the size of a normal

RBC). The nucleus occupy more than 85% of the cell (i.e., very high N:C

ratio) with very smooth chromatin, and 1 to 3 nucleoli could be seen. The

very scant cytoplasm present is usually light to slightly dark blue in color, with

no granules or vacuoles of any type. The blasts compromise about 1-2% of

the total nucleated cells in the BM, and not seen in pb.

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2- The promyelocyte (pro's): these cells are very similar to the blast in

everything except the cytoplasm, where the primary pinkish-red granules

could be prominently seen. These non-specific primary granules, known as

the Azurophilic granules are synthesized in this stage and contain

Myeloperoxidase (MPO) enzyme (the hallmark mark of neutrophilic series).

This enzyme remains active until the mature neutrophil stage. It is the

principal enzyme involved in neutrophil phagocytosis (killing of foreign

bodies, e.g., bacteria). Primary granules are made and lost in this stage

(however the enzyme remains until pmn stage). Other molecules found in

the granules include: proteases, lysozymes, muramidase, etc. Pro’s make up to

5% of total nucleated cells in the BM, and not seen in pb.

3- The myelocyte (myelo's): the cell has a wide range of size (16-24 μ) with

many different criteria than the previous 2 stages. The nucleus is usually

round but could be oval with slightly condensed chromatin, and no nucleoli.

The N/C ratio is smaller than previous 2 (less than 70%). The cytoplasm is

pale gray to pinkish due to the presence of secondary granules (also known as

specific granules), which are made in this stage but unseen due to their very

small size. Secondary granules contain Alkaline Phosphatase, acid

hydrolases, lysozymes, and other proteins, but no MPO. The myelocyte is the

last stage capable of mitosis, i.e., cells in the following stages cannot

proliferate (make more of them selves) but only mature (differentiate) to gain

more specific functional abilities. Myelo’s make up to 20% of BM and not

seen in pb.

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4- The metamyelocyte (meta's): the meta is similar to myelo in a lot of things

except that its nucleus is not round or oval anymore, rather the nucleus takes

a kidney, bean, or peanut shape. The cytoplasm is more pinkish than the

previous stage due to presence of the very tiny secondary granules. Meta’s

make up to 30% of BM and rarely seen in pb (only in stress situation where a

large number of granuloid cells are released in to pb to fight a severe infection,

for instance).

5- The band: the last stage before the fully mature and differentiated neutrophil

(pmn). The nucleus has started to lobulate forming a specific shape of a

“horse shoe” and the filaments which, attach (connect) the different lobes have

not yet, developed. Bands spend about 24 hours in pb to become fully mature

lobulated pmn’s. Bands can make up to 20% of pb differential count.

6- The neutrophil (pmn's): the cell is unique in its possession of a multi-lobed

nucleus. Usually 2-3 lobes could be seen, attached to each other by very fine

filaments (thread like inclusions). The color of cytoplasm is identical to that

of RBC’s with very fine tiny granules all over. Neutrophils make up to 65%

of pb differential count.

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OTHER GRANULOID CELLS

Two granuloid cells exist in normal pb and BM known as: 1- the basophil, and 2- the

eosinophil. These cells rise from the same CFU-GEMM as the neutrophil. The

earliest form to be recognized in the bone marrow is at the PROMYELOCYTE

stage. In other words, the blast cell of all myeloid cells is exactly the same, and

could not be distinguished, from one to another. Basophilic (or eosinophilic)

promyelocytes, (as well as their following stages), are recognized by their

characteristic granules. That is, the presence of cytoplasmic dark blue to black

granules for basophils, and bright red ones for the eosinophilic lineage.

The basophil (baso's)

Similar to mature neutrophil but could be slightly bigger than pmn’s. The very

obvious "hallmark" criterion for its identification is the presence of very large dark

blue-blackish granules all over the cell. Even though, the granules are cytoplasmic,

they might appear as if they are nuclear, as well. Basophils and their tissue

equivalent, the mast cells, are very important mediators of allergic reactions,

especially in the “Immediate Hypersensitivity Reaction”. These cells release

HISTAMINE (a powerful chemotactic factor), Platelet Activating Factor (PAF), and

Arachadonic acid. Basophils are rarely seen in pb or BM having a differential pb

count of about 1%.

The eosinophil (eos)

Eosinophils have large refractive bright pinkish-red granules that are restricted to the

cytoplasm and do not overlap the nucleus (as was the case with baso’s). Eosinophils

are increased in parasitic infections (as an inflammatory response), allergy, and

certain skin diseases. Considerably rare eos are found at about 5% of the normal pb

differential count.

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NEUTROPHIL PHAGOCYTOSIS

The major function of neutrophils is in the defense and killing of foreign invading

organisms (mainly, bacteria). The process is known as phagocytosis and involves

specialized premeditated plan:

1- Motility: also known, as “diapedesis” is the process that allows pmn’s free

migration among tissues and pb. Usually, pmn’s move (migrate) freely in pb,

but in response to chemotactic factors, pmn’s can direct themselves toward the

stimulus. Chemotactic factors are certain compounds such as bacterial

endotoxins, C5a (a complement factor), or Arachadonic acid. Tissue that have

been exposed to bacterial insult, releases such compounds leading to pmn’s

migration to the site of infection.

2- Recognition: antibodies or complement fragments attached to the invading

organism help in identifying the invader (a process known as “opsonization”).

Some opsonins include compounds such as C3b (a complement fragment), Ig

G antibody, or fibronectin.

3- Ingestion: the cytoplasm of pmn’s extends outward (in appendages) known

as “psuedopods” until it fuses together around the foreign body (surrounds it)

forming a “phagosome”.

4- De-granulation: this is the process where pmn’s granules empty their load

(compounds) into the phagosome.

5- Killing: this achieved by the release of cytotoxic compounds such as

superoxide and hydrogen peroxide (aided by the Myeloperoxidase enzyme).

Other lysozymes also, act on degeneration of the bacterial cell by lysing its

cell wall. Finally, the invading cell is gradually lysed and the whole complex

(pmn including the invading bacteria) is disposed.

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MONOCYTIC MATURATION

Monocytes and macrophages (activated form of monocytes found mostly in tissues)

are derived from the same stem cell that granuloid cells (pmn’s, baso’s, and eos) are

derived from, which is the CFU-GEMM (CFU-mix). Similar to neutrophil

maturation, the first identifiable cell morphologically is the blast. However, unlike,

neutrophils, there are only 3 maturational stages (cells) in the monocytic series.

These stages are: 1- the monoblast, 2- the promonocyte, and finally, the mature

monocyte.

1- The monoblast:

Morphologically, the monoblast is undistiguishable from the myeloblast. That is, it is

a very large cell of up to 20 μ in size (about 3 times the size of a normal RBC). The

nucleus occupy more than 85% of the cell (i.e., very high N:C ratio) with very smooth

chromatin, and 1 to 3 nucleoli could be seen. The very scant cytoplasm present is

usually light to slightly dark blue in color, with no granules or vacuoles of any type.

Only CD markers, biological assays, and advanced molecular techniques can

differentiate between monoblast and myeloblast.

2- The promonocyte:

This is similar to the promyelocyte in everything, except they don’t have the typical

primary (reddish pink) granules of the promyelocytes. That is, their shape, size,

chromatin quality, and cytoplasm are very much similar to that of promyelocytes.

They also resemble myeloblasts but they might not have nucleoli.

3- The monocyte:

The monocyte ranges in size from 12 to 22 μ but appear large on stained blood films.

They look larger than neutrophils because they strongly adhere to the slide surface.

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The nucleus is very variable in shape where it could be round, horse-shoe, or

lobulated, with possible convolutions and/or folding of the chromatin. The cytoplasm

is irregular in shape with dull, faded, grayish-blue color and very fine granules. The

cytoplasm could have some projections (psuedopods) and may contain vacuoles.

Monocytes constitute 2-10% of pb differential count.

The main function of monocytes is phagocytosis of invading organisms, even though

they are a little slower and less efficient than neutrophils. Furthermore, monocytes

are involved in T cell mediated immunity. Monocytes can live up to 3 days but tissue

macrophages (i.e., the monocytes that got localized inside the tissues) can live for

several months in the tissues.

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