Formation of blood cells (Haemopoiesis)

1. Where blood is made?

Haemopoietic cells (those which produce blood) first appear in the yolk sac of the 2-
week embryo.

By 8 weeks, blood making has become established in the liver of the embryo, and by
12-16 weeks the liver has become the major site of blood cell formation. It remains
an active haemopoietic site until a few weeks before birth. The spleen is also active
during this period, particularly in the production of lymphoid cells, and the fetal
thymus is a transient site for some lymphocytes.

The highly cellular bone marrow becomes an active blood making site from about 20
weeks’ gestation and gradually increases its activity until it becomes the major site
of production about 10 weeks later.

At birth, active blood making red marrow occupies the entire capacity of the bones
and continues to do so for the first 2-3 years after birth.

The red marrow is then very gradually replaced by inactive, fatty, yellow, lymphoid
marrow. The latter begins to develop in the shafts of the long bones and continues
until, by 20-22 years, red marrow is present only in the upper ends of the femur and
humerus and in the flat bones of the sternum, ribs, cranium, pelvis and vertebrae.
However, because of the growth in body and bone size that has occurred during this
period, the total amount of active red marrow (approximately 1000-1500 g) is nearly
identical in the child and the adult.

Adult red marrow has a large reserve capacity for cell production. In childhood and
adulthood, it is possible for blood making sites outside marrow, such as the liver, to
become active if there is excessive demand as, for example, in severe hemolytic
anemia or following hemorrhage.

In old age, red marrow sites are slowly replaced with yellow, inactive marrow.

Red marrow forms all types of blood cell and is also active in the destruction of red
blood cells.

Red marrow is, therefore, one of the largest and most active organs of the human
body, approaching the size of the liver in overall mass although as mentioned it is
distributed in various parts of the body.

About two-thirds of its mass functions in white cell production (leucopoiesis), and
one-third in red cell production (erythropoiesis). However, as we have already seen
there are approximately 700 times as many red cells as white cells in peripheral
blood. This apparent anomaly reflects the shorter life span and hence greater
turnover of the white blood cells in comparison with the red blood cells.

1
It is now generally accepted that all blood cells are made from a relatively few
'uncommitted' cells which are capable of mitosis and of differentiation into
'committed' precursors of each of the main types of blood cell.

The diagram on the left shows the formation of the different types of blood cells from a
common source the stem cell

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3
 Red blood cells (erythrocytes)

The production of red blood cells is referred to as erythropoiesis.

Mature red blood cells develop from haemocytoblasts. This development takes about
7 days and involves three to four mitotic cell divisions, so that each stem cell gives
rise to 8 or 16 cells.

The various cell types in erythrocyte development are characterised by

  the gradual appearance of haemoglobin and disappearance of ribonucleic acid

(RNA) in the cell
  the progressive degeneration of the cell's nucleus which is eventually extruded
from the cell
  the gradual loss of cytoplasmic organelles, for example mitochondria
  a gradual reduction in cell size

The young red cell is called a reticulocyte because of a network of ribonucleic acid
(reticulum) present in its cytoplasm. As the red cell matures the reticulum
disappears. Between 2 and 6% of a new-born baby's circulating red cells are
reticulocytes, but this reduces to less than 2% in the healthy adult. However, the
reticulocyte count increases considerably in conditions in which rapid erythropoiesis
occurs, for example following haemorrhage or acute haemolysis of red cells. A
reticulocyte normally takes about 4 days to mature into an erythrocyte.

In health, erythropoiesis is regulated so that the number of circulating erythrocytes

is maintained within a narrow range. Normally, a little less than l% of the body's
total red blood cells are produced per day and these replace an equivalent number
that have reached the end of their life span. However that still represents a huge
200,000,000,000 cells

Erythropoiesis is stimulated by hypoxia (lack of oxygen). However, oxygen lack does

not act directly on the haemopoietic tissues but instead stimulates the production of
a hormone, erythropoietin. This hormone then stimulates haemopoietic tissues to
produce red cells.

Dietary element and their role in red blood cell production

Protein is required to make red blood cell proteins and also for the globin part of
haemoglobin

Vitamin B6 the role is not clear but deficiency has occasionally been associated with
anaemia

Vitamin B12 and folic acid are needed for DNA synthesis and are essential in the
process of red blood cell formation

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Vitamin C is required for folate metabolism and also facilitates the absorption of
iron. Extremely low levels of Vitamin C are needed before any problems occur.
Anaemia caused by lack of Vitamin C (scurvy) is now extremely rare

Iron required for the haem part of haemoglobin

Copper and Cobalt There is some evidence that these two trace minerals are
essential for the production of red blood cells in other animals but not in
humans

Monocytes

Monocytes are produced in the bone marrow, developing from nucleated precursors,
the monoblast and promonocyte. Mature cells have a life in blood of approximately
3-8 hours and, like granulocytes, there is a circulating and marginating pool.

Monocytes are actively phagocytic (engulf other cells) and, on migration into the
tissues, they mature into larger cells called macrophages (Derives from the Ancient
Greek: macro = big, phage = eat), which can survive in the tissues for long periods.
These cells form the mononuclear phagocytic cells of the mononuclear phagocytic
system (reticuloendothelial system) in bone marrow, liver, spleen and lymph nodes.
Tissue macrophages (sometimes called histiocytes) respond more slowly than
neutrophils to chemotactic stimuli. They engulf and destroy bacteria, protozoa, dead
cells and foreign matter. They also function as modulators of the immune response
by processing antigen structure and facilitating the concentration of antigen at the
lymphocyte's surface. This function is essential in order that full antigenic stimulation
of both T and B lymphocytes can take place.

Granulocytes

As already mentioned granulocytes is the collective name given to three types of

white blood cell. Namely these are neutrophils, basophils and eosinophils.

In terms of their formation (granulopoiesis) they all derive from the same type of
committed stem cells called myeloblasts. After birth and into adulthood
granulopoiesis occurs in the red marrow.

The process of producing granulocytes is characterised by the progressive

condensation and lobulation of the nucleus, loss of RNA and other cytoplasmic
organelles, for example mitochondria, and the development of cytoplasmic granules
in the cells involved.

The development of a polymorph nuclear leukocyte makes take a fortnight, but this
time can be considerably reduced when there is increased demand, as, for example,
in bacterial infection. The red marrow also contains a large reserve pool of mature
granulocytes so that for every circulating cell there may be 50-100 cells in the
marrow.

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Mature cells pass actively through the endothelial lining of the marrow sinusoid into
the circulation. In the circulation, about half the granulocytes adhere closely to the
internal surface of the blood vessels. These are called marginating cells and are not
normally included in the white cell count. The other half circulate in the blood and
exchange with the marginating population.

Within 7 hours, half the granulocytes will have left the circulation in response to
specific requirements for these cells in the tissues. Once a granulocyte has left the
blood it does not return. It may survive in the tissues for 4 or 5 days, or less,
depending on the conditions it meets.

The turnover of granulocytes is, therefore, very high. Dead cells are eliminated from
the body in faeces and respiratory secretions and are also destroyed by tissue
macrophages (monocytes).

No precise mechanisms for the control of granulocyte production have, so far, been
found. However, in health, the count remains relatively constant so it is likely that
homeostatic control mechanisms operate.

Lymphocytes

Lymphocytes are round cells containing large round nuclei. The cytoplasm stains pale
blue and appears non-granular under light microscopy. However, some cytoplasmic
granules and organelles are present.

Morphologically, lymphocytes can be divided into two groups: the more numerous
small lymphocytes, with a diameter of 7-10 m; and large lymphocytes, which have
a diameter of 10-14 m. Lymphocytes are produced in bone marrow from primitive
precursors, the lymphoblast and prolymphocytes. Immature cells migrate to the
thymus and other lymphoid tissues, including that found in bone marrow, and
undergo further division, processing and maturation.

Platelets

Platelets are produced in bone marrow by a process known as thrombopoiesis. They

are formed in the cytoplasm of a very large cell, the megakaryocyte. The cytoplasm
of the megakaryocyte fragments at the edge of the cell. This is called platelet
budding. Megakaryocytes mature in about 10 days, from a large stem cell, the
megakaryoblast.

It is likely that there are thrombopoietic feedback mechanisms as the platelet count
remains fairly constant in health, and platelet production is reduced following an
infusion of platelets and increased following removal of platelets. However, these
feedback mechanisms have not been discovered yet.

6
 This looping diagrammatic animation
shows the process of platelet formation
from a megakaryocyte

At any one time, about two-thirds of the body's platelets are circulating in the blood
and one-third are pooled in the spleen. There is constant exchange between the two
populations. The life span of platelets is between 8 and 12 days. They are destroyed
by macrophages, mainly in the spleen and also in the liver.

Blood cell development in adults

All blood cells develop from pluripotent stem cells that are found in the red bone marrow.
Stem cells make up 10% of cord blood cells and <1% of all adult blood cells. Stem cells
are able to proliferate as well as differentiate into the different types of blood cells. They
are also able to renew themselves.

The pluripotent stem cell is the progenitor

Of the two multipotential stem cell lines: the myeloid and the lymphoid lines. Myeloid
stem cells are precursors of granulocytes, monocyte, RBCs and platelets; lymphoid stem
cells are precursor of lymphocytes.

Myeloid cell line

The myeloid stem cell becomes the CFU-GEMM (colony forming unit, granulocyte-
erythrocyte-monocyte-megakaryocyte).

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CFU terminology comes from tissue culture of cells where marrow cells were injected
and colonies of cells would appear.

CFU-GEMM leads to formation of:

 BFU (burst forming unit erythroid)

 CFU-Meg (megakaryocyte)
 CFU-GM (granulocyte, monocyte/macrophage)
o CFU-G
o CFU-M
o CFU-Eo (eosinophil)
o CFU-Bas (basophil)

Using routine hematology laboratory analysis, we cannot identify the cells up to this
point by morphology alone. They are identified by surface receptor analysis.

All this formation and development of cells is under control of growth factors and
inhibitors, and the microenvironment.

 CSF - colony stimulating factors function at various stages of development of

blood cells. There are several CSFs, each stimulating the development of different
types of cells:
 Interleukins - protein molecules that work with the CSFs to stimulate particular
cell lines to proliferate and differentiate. There are seven interleukins that relate to
hematology. Interleukins are special types of cytokines (a generic term for soluble
molecules that mediate interactions between cells)
 Erythropoietin – (EPO) the hormone produced by kidney to stimulate RBC
production.

Lymphoid cell line

Cells that develop along this line become the pre-B lymphocyte and pre-T lymphocyte
(prothymocyte) cells.

Pools of hematopoietic activity (where are all these cells?)

1. Bone marrow

Stem cell pool - comprised of multipotential stem cells and unipotential committed
colony-forming units (CFUs), all of which are morphologically unidentifiable.

Proliferating pool - cells are capable of DNA synthesis and are undergoing mitosis.

Storage pool - mature cells that are stored for later release into the peripheral blood and
cells that are maturing. They are no longer capable of mitosis.

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2. Peripheral blood

Circulating pool - functioning cells in circulation, in transit to tissues. The blood we draw
for evaluation comes from this pool. The circulating pool is found more towards the
center of the tubular blood vessel.

Marginating pool – Primarily a term used for white blood cells. The cells are adhered to
walls of blood vessels and are ready to move through into the tissues (diapedesis). There
is constant movement between the circulating and marginating pools. At a given time the
ratio of cells in the circulating pool to the cells in the marginating pool is 50:50.
Neutrophils move freely between the two pools.

Neutrophils

Differentiation, proliferation and maturation takes place in the red bone marrow.

Developmental stages:

 myeloblast
 promyelocyte
 myelocyte
 metamyelocyte
 band
 segmented neutrophil

 Proliferative stages include the first three stages. The myeloblasts, promyelocytes and
myelocytes undergo mitosis as they mature. By the time a cell has reached the myelocyte
stage it has undergone 4 or 5 cell divisions.

Maturation & storage stages includes the last three stages.

General maturational changes that can be seen are:

 Overall cell size generally decreases as the cell matures.
 The nucleus decreases in size and starts to indent at the metamyelocyte stage and
is segmented into up to 5 lobes at the segmented neutrophil stage.
 The nuclear chromatin pattern changes fine to coarse as the cell matures. The
coarseness indicates mitotic inactivity (no more mitosis).
 Nucleoli disappear by the myelocyte stage. Nucleoli are composed of RNA and
are associated

Monocytes/Macrophages

These two cells are the same cell type; they are monocytes in the blood, and become
macrophages when they migrate into the tissue. Monocytes comprise 3-7% of circulating
WBCs (0.6 x 109/L)

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Developmental stages are

 monoblast
 promonocyte
 monocyte; macrophage in tissue

Macrophages may live in tissue for months, even years.

Maturational changes of the monocyte

 The monoblast is similar in appearance to myeloblast.

 The Promonocyte largest of three stages.
 The N: C ratio decreases from 4:1 to 2:1 or 1:1 as the cell matures.
 The nuclear shape goes from oval or folded to lobular and irregular.
 The nucleoli disappear as the cell matures.
 The chromatin pattern goes from fine to slightly more coarse with a lacy
appearance.
 A mature monocyte has an abundance of cytoplasm.
 Cytoplasmic color goes from blue to blue-grey as the cell matures.
 There are no significant obvious granules in the cytoplasm which has a "ground
glass" appearance in the mature stage.
 There are often vacuoles in the cytoplasm, particularly in the mature monocytes.
 Mature monocytes are the largest peripheral WBC most of the time.

Macrophage appearance

 Normally seen only in tissue.

 Larger than monocytes.
 Round nucleus, eccentrically placed.
 Large amount of cytoplasm containing vacuoles.

Lymphocytes

Adult normal value of lymphocytes is 20-40% (1500-3000/uL). Newborns typically have

more lymphocytes than neutrophils (16-90% of circulating WBCs).

There are two general types of lymphocytes: B cells and T cells. B cells don’t live as long
as T cells; B cells live a few days while T cells can live up to 4 years with 1% living up to
20 years.

The developmental stages of all lymphocytes can be divided into antigen independent and
the antigen dependent stages.

1. Antigen independent developmental stages

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  A stem cell in red bone marrow differentiates into the lymphoid stem cell.
 The lymphoid stem cells develop into the pre-B cell in the bone marrow and the
pre-T (prothymocyte) in the thymus (bone marrow and thymus are called primary
lymphoid tissue). "B" comes from bursa equivalent, bone marrow and intestinal
lymphoid tissue thought to be the equivalent of bird organ called bursa of
Fabricius, a saclike structure attached to the cloaca where lymphocytes become
immunocompetent B cells). "T" comes the name of a surface antigen found on
mouse T cells called "theta."
 The pre-B cell & prothymocyte give rise to

 lymphoblast
 prolymphocyte
 mature immunocompetent lymphocytes
(small lymphocytes)
 effector cells and
memory cells

Most cells produced in primary tissue die but a small percentage migrate to the secondary
lymphoid tissue. Secondary lymphoid tissue consists of lymph nodes, spleen, Gut
Associated Lymph Tissue (GALT) in the walls of the ileum, and tonsils Proliferation of T
and B cells in the secondary tissue is dependent on antigenic stimulation.

Lymphocytes travel between blood and lymphoid tissue; return to blood via lymphatic
vessels. There are more T cells than B cells.

Maturational characteristics of small lymphocytes

 Overall cell size decreases as the cell matures.

 N:C ratio stays about the same 4:1.
 Nuclear shape is round in small lymphocytes.
 Nucleoli disappear as the cell matures.
 The chromatin pattern becomes more coarse as the cell matures.
 The color of cytoplasm goes from darker blue to lighter blue.
 There are essentially no granules but may seen a few azurophilic granules in
prolymphocytes and small lymphocytes.

2. Blast transformation (antigen-dependent lymphopoiesis)

During development into immunocompetent T and B lymphocytes, the cells acquire

specific antigen receptors (without exposure to these antigens), committing them to
antigen specificity. Subsequent contact with their respective antigens begins a complex
series of events known as blast transformation.

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Blast transformation leads to cloning of lymphocytes that will take part in the immune
response to the invading antigen. This usually occurs in the lymph nodes. The cells
change in appearance. These transformed cells are first called variant lymphocytes, then
immunoblasts and finally differentiate into effector cells or into memory cells.

Both T or B cells go through these changes. The only way to tell the difference is by
doing cell surface marker studies, using monoclonal antibodies, etc.

The variant lymphocytes may be seen in the peripheral blood during viral infections.

The immunoblasts and effector cells are usually only in the lymph nodes and other
secondary lymphoid tissue. Under intense stimulation of the immune system however,
the immunoblasts and plasma cells (B effector cells) may be seen in the peripheral blood.

A variant lymphocyte is a lymphocyte that is not normal but represents an immune

response e.g. mononucleosis. Our book calls them reactive lymphocytes The term variant
lymphocyte will include all transitional stages occurring after the mature
immunocompetent lymph is stimulated by antigens. Variant lymphocyte will replace all
other terms: Downey cells, atypical, reactive, transformed lymphocytoid or plasmacytoid
lymphocytes, virocytes, stimulated, activated lymphocytes.

Characteristics of variant lymphocytes:

 Increased size
 Enlarged nucleus
 Nucleus may be lobulated or monocytoid, rectangular or flat (2-D)
 Chromatin patterns vary from fine to coarse (fine indicates increased DNA
synthesis and mitosis)
 Nucleoli may be present indicating RNA synthesis, cell division and proliferation)
 Abundant cytoplasm, frequently vacuolated
 Gray to blue cytoplasm (blue indicates RNA and ribosomes, protein production)
 Granules may be present
 Variant lymphocytes may be confused with lymphoblasts.

 Characteristic of the effector plasma cell (plasmacyte):

 Deeper blue cytoplasm

 Eccentric nucleus
 Perinuclear clear zone indicating the area of the Golgi apparatus
 Coarse chromatin pattern

Null cells

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Null cells are lymphocytes that have neither T or B cell markers but do have some of the
common lymph markers. Null cells cannot be identified morphologically. Three kinds of
null cells:

 Killer cells (K cells) - lymphocytes that are activated by an antibody bound to a

cell (opsonization). These cells are involved with an antibody-dependent cell-
mediated cytotoxicity reaction (ADCC).
 Natural killer cells (NK cells) are not dependent on an antibody to recognize
foreign cells. They constantly survey the body for cells that have any surface
alterations like cancer cells, some cells of the embryo, some bone marrow cells
and thymus cells and microbe-infected cells.
 Lymphokine-activated killer cells (LAK cells) When activated by IL-2 they lyse
tumor target cells that are resistant to NK cells.

Function of monocytes and macrophages

Monocytes are found in peripheral blood; macrophages are found in tissue.

There is not a large reserve pool of monocytes in the bone marrow. Monocytes are found
both in the circulating and the marginal peripheral blood pools. Stimulated by growth
factors, monocytes migrate into tissues and are generally called macrophages; some fixed
in connective tissue fibers and other sites and some wandering freely through the
connective tissue.

Macrophages are most numerous in "filter" organs like the spleen, liver, lungs, lymph
nodes collectively known as the mononuclear phagocyte system, (formerly the
reticuloendothelial system) a system that serves as an important body defense mechanism
composed of phagocytic cells.

Special names of macrophages are:

 histiocytes in loose connective tissue.

 Kupffer cells in the liver
 osteoclasts in bone (they absorb and remove bone)
 microglial cells in nervous tissue like the brain
 Langerhans’ cells in the epidermis - they recognize antigens, ingest them and
present them to lymphocytes for eventual destruction
 glomerular mesangial cells in the kidney
 pulmonary alveolar macrophages in the lungs
 macrophages in the spleen and lymph nodes
 monocytes in the blood

Both monocytes and macrophages are phagocytic like neutrophils. Monocyte numbers
increase whenever there is increased cell damage.

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Monocytes are less selective than neutrophils and will ingest more variable material like
old or abnormal erythrocytes.

Monocytes store released iron from lysed erythrocytes. They act as antigen presenters in
that they process ingested material and present the antigen on its (the monocyte’s) surface
to a T-helper (CD4+) lymphocyte. Monocytes also produce and respond to interleukins
and other cytokines involved in the immune response.

Functions of Lymphocytes

The acquired (specific) immune system (vs. non-specific, innate) is comprised of the
humoral and the cell-mediated immune systems. The lymphocytes found in each system
are preprogrammed to respond to a specific antigen. The innate or non-specific immune
system involves skin and other mucosal surfaces, inflammatory response, commensal
bacteria, and phagocytosis.

The acquired (AKA adaptive or specific) immune system involves two systems:

 humoral: humors in medieval physiology were body fluids that determined a

person’s health and temperament. Humors also were proteins that protected
against disease. B lymphocytes are primarily involved with this type of immunity.
 cell-mediated. T lymphocytes are responsible for this type of immunity.

B-lymphocytes are responsible for the humoral response. Upon antigenic encounter they
transform into plasma cells that produce antibodies specific to the antigen. Production of
antibodies is a complex process. Memory B cells are also produced. These memory cells
respond more quickly than the initial response to a future encounter with the same
antigen. Each activated B cell gives rise to many progeny which together are called a
clone of cells because they are all identical.

T-lymphocytes are responsible for the cell-mediated response. After antigenic stimulation
they proliferate and differentiate into memory cells and effector cells. There are at least
two types of effector cells: helper T-cells (CD4+) and cytotoxic T-cells (CD8+). CD =
cluster differentiation markers. They are identifying membrane proteins (antigens). It is
the CD4+ lymphocytes to which HIV has an affinity. The normal ratio of CD4+:CD8+ is
2:1. In HIV-infected individuals the ratio decreases and reverses as the AIDS virus
invades and destroys the CD4+ cells. The ratio is used to monitor the progress of the
disease.

T-helper cells "help" the B-cells produce antibodies by releasing cytokines. They also
affect the action of other cells, such as CD8+. CD8+ cells (cytotoxic T-cells) mediate
destruction of cells infected with pathogens such as viruses, bacterial, protozoa, or fungi;
and are also responsible for rejection in organ transplants. T-helper cells help B-cells
produce antibodies and also influence other T-helpers, T-suppressors and cytotoxic T-
cells. (T suppressors help keep the immune system in control).

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Thrombocytes are also known as platelets. They are metabolically active cell fragments
that are important in hemostasis.

Thrombocytes develop from CFU-GEMM, BFU-M (burst forming unit-megakaryocyte),

and CFU-M (colony forming unit-megakaryocyte).

Megakaryocytopoiesis proceeds through mitotic division of progenitor cells followed by

endoreduplication (mitosis without subsequent division of cytoplasm into identical
daughter cells).

 megakaryoblast
 promegakaryocyte
 megakaryocyte

The megakaryocyte sheds platelets from the cytoplasm. This takes place in bone marrow.
There will be pink areas in the blue cytoplasm signifying platelet development. Increased
granularity is also seen. One megakaryocyte produces an average of 1000 platelets, which
enter the spleen and remain for 2 days. After that they either enter the circulating blood or
remain in the active splenic pool.

The megakaryocyte

 large (up to 160 um) cell

 nucleus is purple with 2 or more lobes
 nucleoli are not visible
 abundant cytoplasm is pinkish-blue with an irregular border

Mature platelet is small (2-4 um)

 anucleate
 cytoplasm is light blue with scattered red-purple granules.

Platelets have specific roles in the coagulation (hemostasis) process. These roles are
dependent on adequate numbers of platelets present and normal platelet function. There
are three specific platelet functions:

1. Maintenance of vascular integrity. Platelets help nurture the endothelial cells lining the
vascular system. The platelets attach to the endothelium and release endothelial growth
factor into the endothelial cells. If platelets are not present in adequate numbers, large
numbers of RBCs can migrate through the endothelial wall and produce petechiae or
purpura in the skin.

2. Platelet plug formation. Exposure to subendothelial connective tissue attracts platelets,

which adhere to the tissue irreversibly (platelet adhesion). The platelets adhere to the

15
injured area as well as to each other to try and stop hemorrhage. Absence of platelet
adhesion can result in bleeding disorders. Once the platelets have adhered to the site of
the injury they change shape to intertwine with each other (platelet aggregation). As the
platelets squeeze together, they release platelet factors (PFs) which are necessary for
completion of the coagulation process.

3. Stabilization of the hemostatic plug by contributing to the process of fibrin formation.

Various clotting factors and enzymes are necessary for successful hemostasis. All of
these factors lead to the generation of large quantities of thrombin on the aggregated
platelet's surface. Thrombin then converts fibrinogen to fibrin, one of the final stages of
clot formation.

Platelets can be evaluated on a peripheral blood smear. Using the same fields you use for
a WBC differential and RBC morphology evaluation, determine the approximate number
of platelets per field. A normal blood smear should have between 8 and 20 platelets per
field in this area of the smear. An platelet estimate is obtained by finding the average
number of platelets in 10-15 fields and multiplying this result by 20,000. A normal
platelet estimate is between 200,000-399,999/microliter.

What’s happening as the cells change in appearance?

The nucleus in immature cells is large because it is physiologically active. The nucleus
contains euchromatin (light, loose, active with nucleoli) if the cell is engaged in mitosis.
The nucleus contains heterochromatin (dark, clumped, inactive) if the cell cannot divide.

Cytoplasm will be rich with RNA (blue) if a lot of protein synthesis is occurring (The
RNA produces protein such as antibodies, cytokines or proteins associated with cell
division). If there is an abundance of protein production, the Golgi Apparatus (part of the
endoplasmic reticulum that prepares the protein for secretion) will be very prominent as a
clear area near the nucleus (perinuclear clear area).

The specific cytoplasmic granules will have various shapes and colors dependent on their
content.

Eosinophils and Basophils

Eosinophils normally comprise about 3% of circulating WBCs.

Basophils normally comprise about 0.5% of the circulating WBCs

The maturation sequence is similar to neutrophils but only 2 or 3 segments form at the
mature stage.

Secondary granules are different color; eosinophils have red granules, basophils have
deep purple to black that may obscure the nucleus and/or cytoplasm. Basophil granules

16
are water soluble and may wash out in poorly fixed smears and appear as empty areas in
the cytoplasm. This gives the cytoplasm a white appearance.

Functions of Granulocytes

Neutrophils

Neutrophils respond to an inflammatory reaction caused by biological, chemical or

physical assault such as heat, cold, excess sunlight, stress, physical trauma. Neutrophils
are attracted to the site of inflammation by chemotaxis, chemical signals sent out by
injured cells or by microbes. They are highly motile and leave through the capillary walls
by diapedesis and migrate to the tissue site. If the inflammatory reaction is severe
enough, initially there may be a neutropenia. This can be followed by a neutrophilia, and
a resulting left shift.

Functional development of neutrophils

1. Myeloblast-essentially a nonfunctional cell.

2. Promyelocyte - primary granules contain myeloperoxidase, an enzyme required for

intracellular microbial killing, and other enzymes.

3. Myelocyte - cell acquires some motility. The secondary granules that begin to be
produced during this stage contain important compounds. Alkaline phosphatase is one of
these compounds. It can be detected using a special stain used to differentiate CML from
leukemoid reactions.

4. Metamyelocytes - cytoplasm contains both primary and secondary granules which

contain compounds necessary to kill and degrade foreign agents (toxic, infectious or non-
self agents); A metamyelocyte still can’t respond to chemotactic factors and can’t initiate
phagocytosis.

5. Band - possesses full motility, active adhesion properties (needed for phagocytosis)
and some phagocytic abilities. They are almost mature neutrophils.

6. Segmented (mature) - the fully functional neutrophil, actively motile and able to
respond to chemotaxis.

The primary function of neutrophils is phagocytosis. Phagocytosis routinely takes place

in the respiratory system, the gastrointestinal system and the urinary system. It is the
process by which cells engulf and disable particles.

Toxic change: appearance of neutrophils when they are active against severe bacterial
infections includes toxic granulation, vacuolization of the cytoplasm, Dohle bodies, and

17
left shift. This is a typical reaction characterized by an increase in digestive enzymes and
digestive vacuoles with an increased RNA activity.

Steps of phagocytosis:

1. Opsonization (Greek = to prepare for dining) Neutrophils cannot efficiently

recognize and attach to most microbes; this process marks the organism for
ingestion by coating the particle with immunoglobulin (antibody) and
complement (a series of proteins that cause disruption of the bacterial membrane).
2. Neutrophil membrane pseudopods envelop the microbe forming a vacuole called
a phagosome within the cytoplasm.
3. The phagosome fuses with lysosomal granules from the neutrophil’s cytoplasm.
The granules release lytic enzymes into the phagosome.
4. The lytic enzymes lead to eventual killing and digestion of the foreign agent. All
these processes require energy that is derived by anaerobic glycolysis (glucose
breakdown).

One of the products formed in the digestion of the foreign particle is hydrogen peroxide,
which is capable of killing microorganisms. Myeloperoxidase, one of the enzymes in the
primary granules, catalyzes a reaction involving H2O2 resulting in a more toxic product.
Myeloperoxidase deficiency is reported to be the most common congenital neutrophil
disorders. However, the condition is fairly benign except for patients with other
problems, like diabetes where fungal infections may occur.

Function of basophils

Basophils play a role in acute allergic reactions. Their granules contain histamine,
heparin and other substances that are released in response to the presence of allergens.
These substances cause increased vascular permeability, smooth muscle spasm,
vasodilation, and the clinical symptoms of an allergic reaction: watery eyes, runny nose,
and difficult breathing.

Histamine is a vasodilator that makes blood vessels more permeable. This effect is
usually seen at inflammatory sites and allows increased cellular movement through the
vessel walls.

Heparin prevents blood clotting. Both histamine and heparin enhance the migration of
leukocytes to the inflamed site.

Function of eosinophils

Eosinophil granules contain enzymes, cytotoxic proteins and cytokine mediators.

Eosinophils are found in high numbers in intestinal and pulmonary mucosae and in the
dermis of the skin. They increase in number when the body is invaded by some parasites
and during allergy attacks. Their granular contents are released to respond to the parasites
and allergens. Eosinophil granular content react with products from basophils, mast cells,

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lymphocytes. For example, eosinophil granules contain histaminase which inactivates the
net effect of released factors is to decrease the inflammatory response and reduce
granulocyte migration into the site of invasion

Monocytes/Macrophages

These two cells are the same cell type; they are monocytes in the blood, and become
macrophages when they migrate into the tissue. Monocytes comprise 3-7% of circulating
WBCs (0.6 x 109/L)

Developmental stages are

 monoblast
 promonocyte
 monocyte; macrophage in tissue

Macrophages may live in tissue for months, even years.

Maturational changes of the monocyte

 The monoblast is similar in appearance to myeloblast.

 The promonocyte largest of three stages.
 The N:C ratio decreases from 4:1 to 2:1 or 1:1 as the cell matures.
 The nuclear shape goes from oval or folded to lobular and irregular.
 The nucleoli disappear as the cell matures.
 The chromatin pattern goes from fine to slightly coarser with a lacy appearance.
 A mature monocyte has an abundance of cytoplasm.
 Cytoplasmic color goes from blue to blue-gray as the cell matures.
 There are no significant obvious granules in the cytoplasm which has a "ground
glass" appearance in the mature stage.
 There are often vacuoles in the cytoplasm, particularly in the mature monocytes.
 Mature monocytes are the largest peripheral WBC most of the time.

Macrophage appearance

 Normally seen only in tissue.

 Larger than monocytes.
 Round nucleus, eccentrically placed.
 Large amount of cytoplasm containing vacuoles.

Special names of macrophages

 histiocytes in loose connective tissue.

 Kupffer cells in the liver
 osteoclasts in bone (they absorb and remove bone)
 microglial cells in nervous tissue like the brain

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 Langerhans’ cells in the epidermis - they recognize antigens, ingest them and
present them to lymphocytes for eventual destruction
 glomerular mesangial cells in the kidney
 pulmonary alveolar macrophages in the lungs
 macrophages in the spleen and lymph nodes
 monocytes in the blood

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