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Hematology 425
Leukopoiesis
Russ Morrison
October 11, 2006
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Leukopoiesis
Leukopoiesis is the development of
WBCs
WBC development (except lymphycytes)
occurs in the same locations as RBCs
(review figure 6-1)
In WBCs the maturation changes are
more unidirectional since with the
exception of neoplasia or myeloid
metaplasia, the spleen and liver do not
participate in WBC formation after birth
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Leukopoiesis
Though one term, erythron, is used to
define RBC production, there is no
corollary for WBC production
WBC production involves complex
populations of cells with different
compartments that they occupy during
their life cycle
Control mechanisms of cellular behavior
are more complex in WBCs than in RBCs
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Leukopoiesis
WBC control mechanisms include interrelations
with adipose tissue, fibroblasts and endothelial
cells
Again, as with RBCs, cellular production takes
place in all marrow space at birth and by the end
of adolescence is found only in the marrow of the
proximal ends of the long bones and in flat bones
such as the skull and sternum. (review fig.6-2)
The inactive marrow of adolescence and
adulthood can revert to active marrow in times of
stress
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Leukopoiesis
WBCs can be divided into categories
based on specific function, site of origin or
morphology
All WBCs exist to defend the body against
nonself agents
This defense is accomplished through
intricate cooperation and communication
among cells
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Leukopoiesis
As a for instance, phagocytes attack and
destroy a wide variety of invading matter
on their own. However, lymphocytes
direct and amplify phagocytic action
through the release of lymphokines (a sort
of bioresponse mediator)
WBCs are divided into granulocytes and
lymphocytes based on differentiation at the
primitive stem cell level (fig. 11-1)
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Leukopoiesis
Lymphocytes are produced in both bone
marrow and lymphoid tissue
Environmental and hormonal stimuli of
lymphocytes are different than those that
control granulocytes and monocytes
Granulocytes (PMNs) function as
destroyers of pyogenic bacteria,
monocyte/macrophages are less
descriminating in their dietary preferences
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Leukopoiesis
Granulocytes contain visible granules and
develop in the bone marrow
Granulocytes are subdivided according to
morphology and according to size/visibility of
granules
Cells containing large, visible granules are called
granulocytes and are further divided into PMNs,
Eos and Basos based on differential staining of
the granules with Romanowsky-based stain
Monocytes contain tiny granules that cause their
cytoplasm to appear grainy with light microscopy
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Leukopoiesis
Microscopic evaluation of WBCs is the basis of
clinical study
Flow cytometry of receptor sites, antigenic
labeling and even functional studies now
contribute to clinical information gathered in the
diagnosis and management of disease
Cell markers have been given alphanumerical
codes (CD1) in which CD stands for cluster
designation, discussed in chapter on flow
cytometry
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Leukopoiesis, Granulocytes
Found in high concentrations in 4
locations called granulocyte pools
1. Bone marrow
2. PB circulation
3. Marginating up against the endothelium
of blood vessels
4. Tissues
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Leukopoiesis, Granulocytes
Bone marrow pool is large and has 3
functions
1. Proliferation
2. Maturation
3. Storage
Cells found in the proliferating
component are myeloblasts,
promyelocytes and myelocytes, all
capable of mitotic division
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Leukopoiesis, Granulocytes
The maturation component of the BM
consists of metamyelocytes and band
forms no longer capable of mitosis but not
yet fully functional
The storage component of the BM consists
of bands and PMNs and holds 25x as many
cells as in the circulating PB
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Leukopoiesis, Granulocytes
Fully mature granulocytes are stimulated
by chemotactic factors and leave the
marrow entering the PB where they
become part of either the marginating pool
of the circulating pool
The marginating pool consists of 50% of
total PB granulocyte levels where the cells
have adhered to blood vessel endothelium
or are engaged in diapedesis (egressing
into tissue through vessel walls)
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Leukopoiesis, Granulocytes
The circulating pool contains the
remaining 50% of PB granulocytes and are
the cells seen and counted in PB
hematologic studies
Granulocytes move freely between
marginating and circulating pools in a bi-
directional flow for a variety of reasons
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Leukopoiesis, Granulocytes
Maturation of the Granulocytic Series
Begins with the pluripotential stem cell
(PSC)
PSC commits its progeny to lymphoid or
bone marrow origin, for reasons unknown,
through the action of growth factors that
are either tyrosine kinase receptors or
cytokines
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Leukopoiesis, Granulocytes
For granulopoiesis, the PSC undergoes
stimulation, mitosis and maturation into a
stem cell that is specific for bone marrow-
derived or myeloid cells
This CFU-GEMM matures into another
stem cell called the CFU-GM
The CFU-GM matures into the earliest cell
of the neutrophilic series, the myeloblast
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Leukopoiesis, Granulocytes
Cell numbers and function is controlled by
complex interaction of humoral factors
such as interleukins and CSFs
CSFs are categorized by the type of cell
stimulated
GM-CSF granulocytes & monocytes/
macrophages
G-CSF granulocytes
M-CSF - monocytes/macrophages
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Leukopoiesis, Granulocytes
CSF specificity is mediated by receptor
sites on precursors and on mature cells
Biologic action of receptors consists of a
ligand specific low-affinity binding chain
and a second, high-affinity chain for
binding and signal transduction.
The second chain interacts with IL-3 and
IL-5 (Chapter 6)
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Neutrophil Maturation - Myeloblast
Cells in the BM proliferation pool take 24-
48 hours for a single cell cycle
Less than 1% of the normal BM
compartment is composed of myeloblasts
Large, 15-20 um in size
Delicate nucleus with prominent nucleoli
Small amount of cytoplasm with rough
endoplasmic reticulum, a developing Golgi
apparatus and an increasing number of
azurophilic granules
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Neutrophil Maturation - Myeloblast
Cytochemical staining shows presence of
myeloperoxidase which is required for
intracellular kills
Killing function is the first to be
operational in the neutrophil cell line
Myeloblast is incapable of motility,
adhesion and phagocytosis and is therefore
nonfunctional
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Neutrophil Maturation -
Promyelocyte
After a few days in the blast stage, the cell
becomes a promyelocyte
1-5% of BM compartment composed of
promyelocytes
Size is variable and may exceed 20 um, so
may be larger than myeloblast
Nuclear chromatin may be delicate or may
show slight clumping
Nuceloli begin to fade
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Neutrophil Maturation -
Promyelocyte
Granules are present throughout the
cytoplasm and on top of the nucleus
Motility may develop by the end of this
stage
Myeloperoxidase is found throughout the
cell which with other enzymes can provide
the peroxidase/superoxide burst capable of
intracellular kill
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Neutrophil Maturation - Myelocyte
Production and accumulation of
neutrophilic granules is characteristic of
the myelocyte
The myelocyte is the last cell of the BM
compartment capable of mitosis
Myelocytes demonstrate morphologic
variability as this development stage lasts
from 4-5 days and cause alterations in the
staining characteristics of the cell
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Neutrophil Maturation - Myelocyte
Smaller in size than the promyelocyte (12-
18 um)
Less than 10% of BM compartment is
made up of myelocytes
Nucleus is round to oval with a flattened
side near the now well-developed Golgi
apparatus
Nuclear chromatin shows clumping
Nucleoli no longer visible
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Neutrophil Maturation - Myelocyte
Secondary granules stain pink causing a
dawn of neutrophilia or pink blush
within the cytoplasm
Compounds such as alkaline phosphatase
begin to concentrate in the cell
The cell acquires some motility
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Neutrophilic Maturation -
Metamyelocyte
The myelocyte becomes a metamyelocyte with
the cessation of all DNA synthesis
Delineator of maturation change is that the
nucleus of the metamyelocyte becomes indented
with clumped chromatin
Complete collection of primary and secondary
granules used to kill and degrade toxic, infectious
or non-self agents
Cell is not yet capable of responding to
chemotactic factors or of initiating phagocytosis
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Neutrophilic Maturation -
Metamyelocyte
13-22 % of BM compartment
10-15 um in size
Not seen in normal PB
Not fully functional, part of the maturation
component of the marrow
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Neutrophilic Maturation - Band
The band is a transitional form that exists
in both the PB and the BM and considered
part of both the maturation and storage
pools
Up to 40% of the WBCs of the BM are
bands
Represents the almost mature neutrophil
having full motility, active adhesion
properties, and some phagocytic ability
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Neutrophilic Maturation - Band
Band forms begin to produce tertiary
granules
Membrane maturity shows changes in
cytoskeleton, surface charge and presence
of receptors for complement
Once entered into the PB, account for less
than 6% of circulating WBCs
10-15 um in size
Found in marginating and circulating poos
of the PB
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Neutrophilic Maturation - PMN
This cells nucleus continues to indent until thin
strands of membrane and heterochromatin form
into segments, hence it is also called a seg
Polymorphonuclear means many-shaped
nucleus, describing the varied nuclear shapes
Cell is completely functional and spend time in
the storage pool of the BM as well as
marginating and circulating pools of the PB
50-70% of circulating WBCs of PB
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Neutrophilic Maturation - PMN
PMNs spend their life performing
phagocytosis and pinocytosis
Phagocytosis involves larger material and
can be observed with light microscopy,
pinocytosis involves small material
(liquids) and is observed with EM
Both of these function can be performed in
the circulation of the blood stream or in the
tissues
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Eosinophil Maturation
Close relative of the PMN whose
secondary granules stain orange-red with
Romanowsky-based stains
Development of PSCs into eosinophils
requires IL-3, IL-5 and GM-CSF and is
inhibited by the presence of interferon
CFU-GEMM to CFU-Eo to myeloblast
Myeloblast to promyelocyte which is
indistinguishable from other
promyelocytes
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Eosinophil Maturation
Myelocyte becomes distinguishable from
neutrophilic line due to presence of large,
round granules containing major basic
protein, which in turn is responsible for the
staining qualities of the eosiniphilic
granules.
Eosinophils spend less than 1 week in the
PB
Large storage capacity of Eos in BM allow
rapid deployment, on demand
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Eosinophil Maturation
When stimulated, Eos leave the marrow
and pass quickly into the tissues
Actively motile, using same migration
paths as neutrophils
Short transit times in PB cause variability
in Eo numbers in the WBC differential
Less than 5% of circulating WBCs
Allergic response may increase numbers of
Eos
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Eosinophil Maturation
Mature Eos may be in band form or
bilobed while nuclei with higher lobe
counts are seldom seen
Slightly larger than PMN at 12-17 um
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Basophil Maturation
Characterized by presence of large, purple
granules
Granules are irregularly shaped, unevely
distributed and deep purple to black when
stained with Romanowsky stains
Maturation from stem cell to mature Baso
is not well defined, but thought to parallel
that of the Eo
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Basophil Maturation
As with Eos, Basos can be classifed as
myelocytes, metamyelocytes, bands and
PMN cells on the basis of nuclear
development
As with Eos, mature cells with more than 2
nuclear lobes are not usually seen
The least common cell in the PB, at less
than 1% of circulating WBCs
Have high-affinity receptors for the Fc
region of IgE
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Monocyte/Macrophage Maturation
Monocyte/Macrophage cells mature from
monoblast to promonocyte to blood monocyte to
free and fixed macrophages, but the mechanism
of commitment is not well understood.
Granular content vary considerably with more
than 50 secretory compounds having been
dentified.
PB monocytes demonstrate morphologic
variability
Aggressive motility and adherence may distort
the monocytes during PB smear preparation
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Monocyte/Macrophage Maturation
Monocyte nucleus is indented or curved
with chromatin that is lacy with small
clumps
Typically the largest cell in the PB
Cytoplasm is filled with minute granules
that produce a cloudy appearance
Cytoplasmic membrane may be irregular,
pseudopods and phagocytic vacuoles may
be evident
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Monocyte/Macrophage Maturation
Described as a transitional cell because it leaves
the BM to enter the PB and then leaves to enter
tissues in response to chemotactic factors
Makes up les than 15% of PB WBC differential
Highly motile and tend to marginate along vessel
walls with a strong tendency to adhere to
surfaces
May be stimulated to undergo diapedesis and
become free macrophages with increased
phagocytic activity
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Monocyte/Macrophage Maturation
Macrophages are large, acively phagocytic
cells with a size of 15-85 um
Pleomorphic in shape, frequently with
pseudopods
Function is phagocytosis
Material ingested is highly variable
Pinocytosis also occurs with items less
than 2 um in size
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Monocyte/Macrophage Maturation
Multistep process of recognition/ attachment,
ingestion, intracellular kill,
digestion/degradation, and exocytosis occurs in
both phagocytosis and pinocytosis.
Monocytes kill any recognizable non-self agents
including dead or dying cells, bacteria, fungi and
viruses.
Play a role in processing antigens for lymphocyte
recognition and stimulation of lymphocyte
transformation.
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Monocyte/Macrophage Maturation
May function as anti-tumor agents by
phagocytic action of nonself cells via
elaboration of tumor necrosis factor and
stimulation of lymphocyte activity
Macrophages are in 2 categories
1. Free found in varying concentrations in
all sites of inflammation and repair,
alveolar spaces and peritoneal and
synovial fluids
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Monocyte/Macrophage Maturation
2. Fixed found in specific concentrations
in specific sites such as the nervous
system (microglial cells), liver (Kupffer
cells), spleen, bone marrow and lymph
nodes
Macrophages are large, 15-80 um, have
ample cytoplasm filled with granules and
often have multiple vacuoles
Nucleus is round to reniform and may
contain 1 or 2 nucleoli
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Lymphocytes
The only human WBCs whose site of
development is not just BM, but also tisues
referred to as primary and secondary
lymphoid organs
In humans, the primary lymphoid organs
are the thymus and bone marrow, the
secondary organs include the spleen,
Peyers patches of the GI tract, the
Waldermyer ring of the tonsils and
adenoids, the lymph nodes and modules
scattered throughout the body
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Lymphocytes
Lymphocytes circulate throughout the body in
both PB and lymph which act as carrier streams
to bring the lymphocytes to sites of activity
Lymphocytes migrate from thoracic duct through
vessel endothelium to lymph nodes to blood
stream and back.
Lymphocytes are categorized in a variety of
ways and may be short-lived or long-lived cells
Lymphocytes may produce antibodies or
lymphokines and have different surface charges,
densities and antigen receptors.
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Lymphocytes - Development
The PSC results in a stem cell for the
lymphoid cell (CFU-L) as a result of
hormonal stimuli
The CFU-L matures in several
environments
Thymus and BM give rise to
lymphocytes, foster differentiation and
are indepentendent of antigenic
stimulation
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Lymphocytes - Development
Cells that develop under the influence of the
thymus are called T cells and have specific
receptors and responses.
B cells develop from the BM and have a different
set of functions and receptors.
The end cell of the B lymphocyte maturation is
the plasma cell
Once the environmental effects of the thymus
and BM have been achieved, lymphocytes
migrate to secondary lymphatic tissues such as
the spleen and tonsils, which act as the main
repositories for already differentiated
lymphocytes.
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Lymphocytes - Development
Cellular interactions for the presentation of
antigen to the cells have a critical role in
priming cells for proliferation and impact
cell maturation, especially T cells. Once
primed, the cells are now responsive to
specific antigens.
Lymphocytes demonstrate lymphoblast,
prolymphocyte and mature lymphocyte
stages when stained with Romanowsky
stains.
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Lymphocytes - Development
Lymphocyte % in the PB varies,
depending on age.
Children under the age of 4 have a higher
proportion of lymphocytes in the PB than
do adults
Lymphocytes are the second most common
WBC of the PB making up 20-40% of
WBCs.
20-35% of circulating lymphocytes are B
cells
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Lymphocytes - Maturation
Lymphoblast to prolymphocyte
Lymphoblast is small, 10-18 um
Round to oval nucleus
Loose chromatin with one or more active
nucleoli
Scanty cytoplasm
Prolymphocyte difficult to distinguish,
subtle changes, more clumped chromatin,
lessening nucleolar priminence, change in
thickness of the nuclear membrane
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Lymphocytes - Maturation
Prolymphocyte to Lymphocyte
Lymphocytes vary mostly by size
1. Small 9 um in diameter, non-dividing
or resting
2. Medium 11-14 um, non-dividing
3. Large 15 um, more generous cytoplasm
that is deep blue when stained
Morphologic variants (table 11-2)
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Lymphocytes Immunologic
Differentiation
Lymphocytes may be classified by
immunologic function
B Cells
1. Possess cytoplasmic IG concentrations of IgD
and IgM
2. Some membrane receptors are apparent
3. The fully committed B lymphocyte is the
plasma cell
4. Demonstrate class I and class II human
leukocyte antigens (HLA-A, HLA-B, HLA-C
and HLA-D, HLA-DR)
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Lymphocytes Immunologic
Differentiation
T Cells
1. The primitive T cell, CFU-L, travels to the
thymus
2. Acquires a transferrin receptor that is specific
to proliferation
3. Mature T cells lose all precursor markers and
have an active helper or suppressor function
4. T cells are further differentiated through
presence or absence of HLA-D antigens
5. T cells possess HLA-A, HLA-B and HLA-C
class I antigens
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Lymphocytes - Activity
The main function of the lymphocyte is to
regulate immune function
If foreign material is completely engulfed,
degraded and disposed of by phagocytes, no
immune response occurs
If digestion is incomplete, antigenic fragments
are transported to lymph nodes
In the lymph node the antigen is fixed to the
exterior surface and brought into the lysozymes
of the macrophage
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Lymphocytes - Activity
The antigen is processed and once that
occurs, proliferation occurs
Development of clones of antigen-specific
B lymphocytes and cytotoxic T cells
begins
Activity that accompanies clonal
expansion required for antigen removal
can be seen in the morphology of cells
called reactive lymphocytes