This document provides an overview of myeloid leucopoiesis, or the formation of white blood cells from myeloid progenitor cells. It discusses the sites where white blood cell formation occurs, the growth factors involved in differentiation and proliferation, and the maturation process for granulocytes and monocytes. The key functions of neutrophils, eosinophils, basophils and monocytes/macrophages are also summarized, including their roles in phagocytosis and the innate immune response.

1. Normal Leucopoiesis
Myeloid

2. Learning outcomes
• Describe the formation of white blood cells.
• Know the sites of Myeloid Leucopoiesis.
• Overview of Phagocytosis.
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Topics – Myeloid Series
1. Sites of leucopoiesis
2. Leucopoietic growth factors
3. Maturation of leucocytes
4. Function of granulocytes and monocytes

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Leucopoiesis
Cellular differentiation, proliferation and
maturation of leucocytes takes place in
haemopoietic tissue and organs.
Leucopoiesis begins in the foetus and
continues throughout life.

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Manual Differential
Perform a 100-cell WBC differential count
Start from a position where the cells do not overlap.
If too thick leucocytes will be distorted by red cells.

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Absolute WBC Counts
% differential counts should be converted to absolute values
to obtain accurate values. See examples below
Example 1
Total WBC = 10.0 x 109/l
% Neutrophil = 50 %
Absolute count = 50 x 10.0 = 5.0 x 109/l
100
In this case 50% Neutrophils is a normal absolute count for an adult.
Example 2
Total WBC = 2.6 x 109/l
% Neutrophil = 50%
Absolute count = 50 x 2.6 = 1.3 x 109/l
100
In this case 50% Neutrophils is an abnormal low absolute count for an adult.

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Nucleated red blood cells should be recorded as number
/100 WBC. A correction should then be applied to the
Total WBC and new absolute values calculated
( 1000)
Corrected WBC = Machine WBC x 100
(100 + Count of NRBC‘s)
Formulae for correcting Total WBC
Example
120 NRBCs/100 WBC
Machine WBC = 20.0 x 109/l
Corrected WBC = 20.0 x 100
100 + 120
Corrected WBC = 9.1 x 109/l

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Sites of Myeloid Leucopoiesis
Foetus - 5 – 9 months
Infants – all bones
Adult - Vertebrae, ribs,
sternum, skull, sacrum,
pelvis, ends of femur
Foetus - 2 – 9 months
Adult - extramedullary
Foetus - 2 – 9 months
Adult - extramedullary

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Granulopoietic Growth Factors
Act on stromal cells
Interleukin-1 (IL-1)
Tissue necrosis factor (TNF)
Act on pluripotential stem cells
Stem cell factor (SCF)
Flt ligand (Ftl-L)
Act on multipotential progenitor cells
Interleukin-3 (IL-3) , Interleukin-6 , Interleukin-11
Granulocyte/macrophage colony stimulating factor (GM-CSF)
Act on committed progenitor cells
Granulocyte colony stimulating factor (G-CSF)
Macrophage colony stimulating factor (M-CSF
Interleukin-5 (eosinophil colony stimulating factor) (IL-5)
CFU = Colony Forming Unit (GEMM = granulocyte,erythrocyte,monocyte,macrophage)
BFU = Burst Forming Unit

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Granulopoietic Growth Factors
CFU = Colony Forming Unit (GEMM = granulocyte,erythrocyte,monocyte,macrophage)
BFU = Burst Forming Unit
• The growth factors stimulate proliferation,
differentiation and also affect the function of the
mature cells they act on.
• The increase production of phagocytes in response
to an infection is induced by increased production of
growth factors from stromal cells and T-lymphocytes,
stimulated by endotoxin, IL-1 or tumour necrosis
factor (TNF)

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Granulopoiesis- progenitor cells
Stem cell factor
CFU GEMM IL-3
CFUB
GM-CSF
CFUEo
CFUGMEo
CFUGM
CFUG
CFUM
CFUM CFUG
IL-5

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Overview of Phagocytosis
Chemotaxis (cell mobilization and migration)
The phagocyte is attracted to micro-organism or the site of inflammation
by chemotactic substances released from damaged tissues or by complement
and also by the interaction of leucocyte adhesion molecules with ligands on
the damaged tissue.
Phagocytosis
The foreign material (eg bacteria) or dead or damaged cells of the host are
phagocytosed. Recognition of the foreign particle is aided by opsonization
with immunoglobulin or complement because phagocytes have Fc and C3b
receptors.
Chemokines
Chemokines are chemotatic cytokines- two main classes
CXC(a) – pro-inflammatory cytokines which mainly act on neutrophils
CC(b)- act on monocytes, basophils, eosinophils and natural killer (NK) cells
(recruit appropriate cells to the site of inflammation, etc)

19. • CXC(a): A chemokine super family in which
there are four conserved cystine (C ) residues
where X is any amino acid.
• CC chemokine: The CC chemokine (or β-
chemokine) proteins have two adjacent
cysteines (amino acids), near their amino
terminus. There have been at least 27 distinct
members of this subgroup reported for
mammals, called CC chemokine ligands
(CCL)-1 to -28; CCL10 is the same as CCL9.
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Overview of Phagocytosis
Killing and Digestion
Oxygen dependent pathway:
Superoxide, hydrogen peroxide and other activated oxygen species
are generated from oxygen and reduced NADPH.
Oxygen independent pathways:
The non-oxidative microbicidal mechanism involves a fall in pH within
phagocytic vacuoles into which lysosomal enzymes are released.
An additional factor, lactoferrin - an iron binding protein present in
neutrophil granules is bacteriostatic by depriving bacteria of iron.

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Figure 10 L'adhésion de bactéries via des récepteurs

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Phagocytosis
Start
Finish
neutrophil
Primary
granules
Secondary
granules
Chemostaxis

23. Phagocytosis
A. Attachment via
receptors
– FcR, complement R,
scavenger R, Toll-
like R
B. Pseudopod
extension
C. Phagosome
formation
D. Granule fusion and
Phagolysosome
A
B
C
D

24. Phagocytosis and intracellular destruction of microbes. Microbes may be ingested by different membrane receptors of
phagocytes; some directly bind microbes, and others bind opsonized microbes. (Note that the Mac-1 integrin binds
microbes opsonized with complement proteins, not shown.) The microbes are internalized into phagosomes, which fuse
with lysosomes to form phagolysosomes, where the microbes are killed by reactive oxygen and nitrogen species and
proteolytic enzymes. iNOS, inducible nitric oxide synthase; NO, nitric oxide; ROS, reactive oxygen species.

25. Nitric oxide-dependent
killing
• Bacteria binds to
macrophage
• Production of TNF-
alpha
• Upregulates iNOS
• Release of NO
• NO is toxic to infected
cells in vicinity of
macrophage
Macrophage
IFN-gamma
TNF
O2 + L-arginine NO + citrulline
NO synthetase
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Complement and Immunoglobulins
Micro organism
(antigen)
Complement
Immunoglobulin

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Neutrophils, eosinophils and basophils

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Myeloid Maturation
At myelocyte stage specific granules develop for each cell line.
acted on by different growth factors
Primary granules (Promyelocyte stage)
myeloperoxidase, acid phosphatase and other hydrolases
Secondary granules (Myelocyte stage)
collagenase, lactoferrin and lysozymes
Eosinophils and Basophils granules also contain other specific proteins/chemicals

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Neutrophil Function
Normal bone marrow contains up to 4% myeloblasts and contains more
myeloid cells than erythroid in a ratio of 2:1 – 12:1
In the normal state the bone marrow contains 10-15 times the number of
granulocytes found in the peripheral blood. Called the storage pool. This
allows immediate release of neutrophils into the peripheral blood stream as
a response to infection (stimulated by growth factors/cytokines)
Following release from the bone marrow the neutrophils spend 6 -10 hours
in the circulation before moving into the tissues where they perform their
phagocytic function. They spend about 4-5 days in the tissue before they are
destroyed during defensive action or as the result of senescence.

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Neutrophil Kinetics and Storage
1
4
4
12
16
4
Neutrophils in the storage pool can be immediately released into the blood stream.
When the demand increases more immature forms are released.

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Function of Neutrophils
Adult normal range: 2.5 – 7.5 x 109/l (54 – 64%)
NOTE: Children’s ranges vary with age
Main Funtion: PHAGOCYTOSIS

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Function of Eosinophils
Adult normal range: 0.1 – 0.5 x 109/l (1-3%)
NOTE: Children’s ranges vary with age
They spend 1-8 hours in circulation before migrating
to tissues (mainly in epithelial layers exposed to the
environment - nose, skin, urininary tract) where they
live for several weeks
Eosinophils have multiple functions and contribute to a variety of immune defence
mechanisms. Their major defensive roles is against helminth parasites. They can also
phagocytose bacteria and have the same chemotaxin receptors(IgG and complement)
as neutrophils, plus receptors for IgE and histamine.
They are associated with chronic inflammation and allergic reactions and have
cytotoxic potential to turn against the hosts tissue (eg Asthma, Hayfever)

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Function of Basophils
Normal range: 0.01 – 0.1 x 109/l (0-1%)
They spend 1-8 hours in circulation before
migrating to tissues where they become
?mast cells
Basophil/mast cell function as mediators of inflammatory responses. When IgE
attaches to the receptor on the cell, it is activated to degranulate, releasing
enzymes that are vasoactive, bronchoconstrictive, and chemotactic.
The granules contain histamine and heparin. The release of large numbers of these
granules in anaphylatic shock may cause death to the host.
Basophils also express CD40L a ligand for B-lymphocytes CD40 antigen which in
conjuction with IL-4 can induce IgE synthesis by B-lymphocytes. Thus, have important
role in inducing and maintaining allergic reactions

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Monoblast

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Function of Monocytes
Normal range: 0.2 – 0.8 x 109/l (4-10%)
They spend 20-40 hours in circulation before migrating to tissues where
they transform to macrophages and can live several months or years.
They may assume specific functions in different tissues.
This is known as the mononuclear phagocytic system (recticuloendothelial)

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Tissue
Macrophages
Mononuclear phagocytic system

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Mononuclear phagocytic system
Collection of monocytes and macrophages found in
- Circulating blood monocytes
- Fixed macrophages of the bone marrow, liver, spleen and lymph
nodes
- Free macrophages of the spleen, lymph nodes, lungs, serous
fluid and other tissues.
Functions
1. Phagocytosis
2. Secretion of growth factors which regulate inflammation
and immune response
3. Processing and presenting foreign antigens on human leucocytes
(HLA) molecules to the immune system. (Dendritic cells)

38. Q & A
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References
• Essential Haematology (4th edition)
– by A. Hoffbrand, J. Pettit and P. Moss
Chapter 1 – Blood cell formation (haemopoiesis)
Chapter 9 – The white cells 1: granulocytes and monocytes

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