Hematology 425 Leukopoiesis

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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
  
• Bone marrow
• PB circulation
• Marginating up against the endothelium of blood
  vessels
  
• Tissues

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Leukopoiesis, Granulocytes

• Bone marrow pool is large and has 3 functions
• Proliferation
• Maturation
• 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/degradati
  on, 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
• 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

• 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
• Small 9 um in diameter, non-dividing or
  resting
  
• Medium 11-14 um, non-dividing
• 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
• Possess cytoplasmic IG concentrations of IgD and
  IgM
  
• Some membrane receptors are apparent
• The fully committed B lymphocyte is the plasma
  cell
  
• 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
• The primitive T cell, CFU-L, travels to the
  thymus
  
• Acquires a transferrin receptor that is specific
  to proliferation
  
• Mature T cells lose all precursor markers and
  have an active helper or suppressor function
  
• T cells are further differentiated through
  presence or absence of HLA-D antigens
  
• 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