Composition, Formation

and Function of Blood

1
Objectives
Upon completion of this portion the student will be able to:
 Explain the composition of blood
 Describe the morphology and functions of the formed elements
of blood
 Discuss the functions of plasma
 Define hemopoiesis and explain the process of blood cell origin
and development
 Indicate the sites of hemopoiesis in infancy, childhood and
adulthood
 List at least three hemopoietic growth factors
 Name the cells in the development order that will mature into
erythrocytes, thrombocytes and the five leukocytes

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Objectives cont’d
 Discuss how hemopoiesis is regulated
 Describe the morphology of the red blood cell, white blood cell, and
platelet precursors
 Define extramedullary hemopoiesis
 Differentiate between intramedulary and extramedulary hemopoiesis
 Define erythropoiesis
 Explain how erythropiesis is regulated and list the effects of the hormone
erythropoietin on erythropoiesis
 Define megaloblastic erythropoiesis
 Define ineffective erythropoiesis
 Define myeloid erythroid ratio

3
 Outline
 Introduction
 Composition of Blood
 Characteristics of Blood
 Formation of blood cells
 Hemopoiesis
 The Hemopoietic Microenvironment
 Regulation of Hemopoiesis
 Maturational characteristics of
hemopoietic cells

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Introduction
Definition of Hematology
 Greek term

◦ Haima means blood

◦ Logos means discourse
 Hence, Hematology is defined as the study of blood and blood-forming
tissues.
 It is the study of the formed elements in the blood
 It encompasses:

◦ the study of blood cells and coagulation

◦ Analyses of concentration, structure and function of cells in blood and
their precursors in the bone marrow

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 Composition of Blood
 Blood
 is the only fluid connective tissue
 constitutes 6-8% of the total body weight
 consists of cells suspended in a fluid called plasma.
 about 45% cells; 55% plasma

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Composition of Blood cont’d

(WBC + platelets)

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 Composition cont’d
 Plasma
 part of the extracellular fluid of blood
 a complex solution of proteins, salts and numerous metabolic
substances
 acts as a transport medium carrying its constituents to
specialized organs of the body.
 Consists of:
 about 91.5% water
 about 8.5% solutes of which about 7% are proteins
 Out of the 7% protein:
 54% albumin
 38% globulins
 7% fibrinogen
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Composition of Plasma
Constituent Percentage of plasma
Water 90-92 %

Protein 6-8%

Inorganic ions <1% (0.9%)

Organic ions <1% (0.5-0.9%)

Plasma Proteins Plasma concentration

Albumin 4.5 %

Globulin 2.5%

Fibrinogen 0.25%

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Formed Elements
 The three main blood cells/formed elements are:
 red blood cells (erythrocytes)
 white blood cells (leucocytes)
 platelets (thrombocytes)

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Erythrocytes (Red Blood
Cells)
 Are the most numerous cells in the blood
 The normal RBC count is approximately 4.5 to 6 million
cells per micro -liter.
 Their primary function is gas exchange.
 carry oxygen from the lungs to the tissues
 return carbon dioxide (CO2), a waste product of
metabolism, from the tissues to the lungs to be
exhaled
 are anucleated cells containing few organelles
 a large proportion of their cytoplasm consists of the iron
containing oxygen transport molecule hemoglobin.

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Erythrocytes cont’d
 shaped like biconcave disks approximately 7 to 8 m in diameter
with a thickness of 1.7-2.4m
 The biconcave disk shape gives red blood cells (RBCs) the
flexibility to squeeze their way through capillaries and other
small blood vessels.
 In stained smears, RBCs look like a circle with a central hole, or
central pallor, which is approximately one-third the diameter of
the cell
 normally survives in the blood stream for approximately 120
days
 after finishing its life span, it is removed by the phagocytic cells
of the reticuloendothelial system, broken down and some of its
constituents re utilized for the formation of new cells.

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Erythrocytes

 Note that the size of the erythrocytes is about the

same as the nucleus of the small resting
lymphocyte.

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Leukocytes (White Blood Cells)
 Leukocytes are :
 a heterogeneous group of nucleated cells
 responsible for the body’s defenses
 transported by the blood to the various tissues where
they exert their physiologic role, e.g. phagocytosis.
 The normal WBC count is ~4,000 to 10,000/L (4.0–10.0 x
103/L)
 Leukocytes are usually divided into:
 Granulocytes, which have specific granules, and
 Agranulocytes, which lack specific granules

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 Leukocytes (White Blood Cells)
 Granulocytes/ Polymorphonuclear leukocytes are divided into:
 Neutrophils (with faintly staining granules),
 Eosinophils (with large reddish or eosinophilic
granules), and
 Basophils (with large dark blue or basophilic
granules).
 Agranulocytes/mononuclear leukocytes are divided into:
 Lymphocytes and
 Monocytes.
 Although they are called white blood cells, leukocytes
predominantly function in tissues.
 They are only in the blood transiently, while they travel to their
site of action.
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Neutrophils
 are the most common type of WBCs in adults
 The segmented neutrophils “segs,” also called
polymorphonuclear neutrophil leukocytes [PMNs or
“polys”]
 are the primary defense against bacterial infection
 Their size ranges from 10-12m in diameter.
 They are capable of amoeboid movement.
 There are 2-5 lobes to their nucleus that stain purple
violet.
 The cytoplasm stains light pink with pinkish dust
like granules.

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Neutrophils cont’d
 Normal range: 2.0-7.5 x 103/l.
 Increased in acute bacterial infections.

Band Neutrphil

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Eosinophils
 Have the same size as neutrophils or may be a bit
larger (12-14m).
 The nucleus:
 is often bilobed with a "spectacle"
arrangement.
 stains a little paler than that of neutrophils.
 Cytoplasm contains many, large, round/oval orange
pink granules.
 They are involved in allergic reactions and in
combating helminthic infections.
 Normal range: 40-400/l.
 Increase in their number (eosinophilia) is associated
with allergic reactions and helminthiasis.

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 Basophils
 Size: 10-12m in diameter.
 are the least common type of leukocytes, normally
≤1% of total WBCs.
 Have a kidney shaped nucleus often obscured by a
mass of large deep purple/dark blue staining
(basophilic) granules.
 The granules contain:

◦ heparin (an anticoagulant),

◦ histamine (a fast vasodilator),
◦ the slow-reacting substance of anaphylaxis
(a slow vasodilator), and other
compounds.
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Basophils cont’d
 involved in immediate hypersensitivity reactions related
to immunoglobulin class E (IgE)
 Normal range: 20-200/l. Basophilia is rare except in
cases of chronic myeloid leukemia.

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Lymphocytes
 are the second most common type of leukocytes in
adults (~20–40% of WBC)
 The average number of lymphocytes in the peripheral
blood is 2500/l.
 The lymphocyte number is higher in children and also
increases with viral infections

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 Lymphocytes cont’d

1. Small Lymphocytes/Resting lymphocytes:

 are usually small (7-10m in diameter)
 has a dark round to oval nucleus, and
 only a rim of pale blue staining cytoplasm
 nucleus is about the same diameter as a
normal erythrocyte & occupies most of the
cell
 are the predominant forms found in the
blood.

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2. Large Lymphocyte
 A small number of lymphocytes in the blood
 Slightly larger than resting lymphocytes, with
reddish purple (azurophilic) granules. This
appearance generally corresponds to natural
killer (NK) cells
 Size: 12-14m in diameter
 Nucleus:
 a little paler than small lymphocytes
 is usually eccentrically placed in the cell
 Cytoplasm:
 Is more plentiful
 stains pale blue and may contain a few
reddish (azurophilic) granules.

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Monocytes
 Are the largest white cells measuring 14-18m in diameter.
 Normally comprise ~2 to 8% of leukocytes
 After 8 to 14 hours in the blood, they enter tissue to become
tissue macrophages (also called histiocytes)
 Cytoplasm:

◦ abundant staining light gray to light blue

◦ finely granular
 Nucleus has very finely granular chromatin and is often
folded, bean shaped, oval, or irregular

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Monocytes cont’d
 Monocytes have two functions:
 Phagocytosis of microorganisms (particularly
fungi and mycobacteria) and debris
 Antigen processing and presentation. In this role, they are
critical in initiation of immune reactions
 Normal range: 700-1500/l.
 Monocytosis is seen in bacterial infections (e.g.,
tuberculosis) and protozoan infections.

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 *Values given are for adults; children tend to have a higher
proportion of lymphocytes. The exact ranges will vary slightly
between different laboratories.
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Platelets (Thrombocytes)
 are small, non nucleated (anucleated), round/oval cells/cell
fragments
 Their size ranges 1-4m in diameter
 The cytoplasm stain pale blue and contain many pink
granules
 They are produced in the bone marrow by fragmentation of
megakaryocytes, which are large and multinucleated cells
 Their primary function is preventing blood loss from
hemorrhage by forming a platelet plug

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 Platelets
 Platelets
have a life span of approximately 10 days.
 Senescent platelets are removed by the spleen
 Normal range: 150-400 x 103 /l.

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Characteristics of Blood
1. Temperature
◦ Roughly 38°C (100.4 °F)
2. Viscosity
◦ Five times that of H2O due to interactions among
dissolved proteins, formed elements, & surrounding
H2O molecules
◦ Sticky, cohesive, and resistant to flow
3. pH
◦ Ranges from 7.35- 7.45, averaging 7.4

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Characteristics of Blood cont’d
4. Volume
 5-6 liters in adult male
 4-5 liters in adult female
 differences between genders reflect differences in
body size
 Blood volume (BV) can be estimated by calculating
7% of the body wt in Kg
 E.g. 75 Kg individual would have a BV of approximately
5.25 liters (~1.4 gallons)
 Hypovolemic = below normal
 Normovolemic = normal
 Hypervolemic = above normal
 Abnormally high BV can place severe stress on the heart

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Function of Blood
 Transportation
 O2 to tissues & CO2 from tissues to lung
 Nutrients from GIT to cells
 Heat and waste products from cells for excretion
 Hormones from endocrine glands to other body cells
 Regulation
 pH
 Temperature
 Osmotic pressure (influence water and ion content of
cells)

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Function of Blood cont’d
Protection
◦ From bleeding (by the clotting mechanism)
◦ Immunity (phagocytes, lymphocytes, antibodies,
complement proteins, etc)

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Formation and Regulation of Blood
Cells production
 Hematopoiesis / Hemopoiesis
◦ is the process of blood cell formation,
differentiation and development
 Origin of Blood cells
◦ There have been two theories
 Monophyletic theory – all blood cells originate from a
single mother cell
 Polyphyletic theory – several mother cells give rise to the
different cell lineages
monophyletic theory is accepted by many hematologists

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Hematopoiesis cont’d
 According to this theory:
 all blood cells (RBC, WBC, PLT) originated from a
Pluripotent stem cell (PSC)
 PSC is the first in a sequence of regular and orderly
steps of cell growth and maturation
 Depending on the conditioning stimuli and mediators
(colony-stimulating factors, erythropoietin,
interleukin, etc.), PSCs mature along
morphologically and functionally diverse lines

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Hematopoiesis cont’d
 PSCs:

◦ Produce other stem cells and self-regenerate

maintaining their original numbers (self renewal),
or
◦ Differentiate into:
 Lymphoid cell line for lymphopoiesis
 Myeloid cell line for myelopoiesis
 is a multipotent stem cell (MSC) capable of
granulopoiesis, erythropoiesis and thrombopoiesis.
 The MSC will first give rise to CFU-GEMM

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 Hemopoiesis cont’d
 In response to specific cytokines, CFU-GEMM
produces erythroid, granulocytic (Eos, Baso, Neut),
Monocyte/macrophage and megakaryotic cells.

 The Lymphoid stem cell (LSC) differentiates into a committed

pre-B and Pre-T cells that from B & T lymphocytes

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37
Stages in hemopoietic cell
development

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 Sites of Hemopoiesis
 The sites of blood cell development follow a definite sequence
from embryonic life to fetal life, to childhood, and to adult life.
 Fetus:

1. Embryonic Yolk sac

 Is the site where mesoblastic phase of hemopoiesis occurs
 Dominates during the first 2-8 weeks of life
 The earliest hemopoietic cells to be produced are the
primitive erythroid precursors (in 2 weeks old embryo)
 Stops at 8 -10 weeks of gestation

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 Sites of Hemopoiesis
cont’d
2. Liver and spleen:
 gradually replace yolk sac
 are the sites where the hepatic phase of hemopoiesis takes
place
 Liver is major site by the 2nd month
 appearance of granulocytes and megakaryocytes.
 Liver and spleen predominate 2-5 months
 Production in the liver tails off within 1-2 weeks of delivery
3. Bone marrow:
 begins in the 4th month
 After 5th month, it is the primary site of hemopoiesis

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Site of hemopoiesis

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Stages/Phases of hemopoiesis in the
embryo and fetus

Months

 Stages of hemopoiesis in the embryo and fetus, indicating the comparative

participation of the chief centers of hematopoiesis and the approximate
times at which the different types of cells make their appearance
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The Bone marrow during infancy,
childhood and adulthood
◦ In infancy
 Red & hematopoietic (active)
◦ During childhood
 Replacement of red marrow with fatty tissue
◦ During Adulthood
 The marrow of the central skeleton (vertebrae, sternum) &
proximal ends of long bones (femurus, humurus) consist of
50% fatty space
 BM cavities in body contain non-hematopoietic fatty
marrow

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Medullary hemopoiesis
 Blood cell production within the bone marrow
(medulla)

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Extramedulary hemopoiesis
 Formation of apparently normal blood cells outside the
confines of the bone marrow mainly in the liver and spleen in
post fetal life is known as Extramedullary Hemopoiesis.
 Occurs when the bone marrow becomes dysfunctional e.g.,
aplastic anemia, infiltration by malignant cells, or over
proliferation of a certain cell (e.g. leukemia)
 When the bone marrow is unable to meet increased
demand for cells, e.g., hemolytic anemia
 If extramedulary hemopoiesis develops, the liver and
spleen are enlarged (hepatosplenomegaly)

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Extramedulary
hemopoiesis

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The Hemopoietic Microenvironment
 Hemopoiesis occurs in a microenvironment in the bone marrow:
◦ in the presence of fat cells, fibroblasts and
macrophages
◦ on a bed of endothelial cells
 The medullary cavities contain:
◦ vascular spaces (sinuses)
◦ hematopoietic cells , and
◦ specialized stromal cells of various types.
◦ All the cells form a complex microenvironment, with
numerous intricate and interdependent relationships
between stromal cells and hematopoietic cells (see
fig).

47
Hemopoietic Microenvironment
cont’d
 anextracellular matrix of fibronectin, collagen and laminin
combines with these cells to provide a setting in which stem
cells can grow and divide.

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Bone Marrow Microenvironment
cont’d
 Hemopoietic Cords (parenchyma) are the extravascular
portions of the bone marrow and the site of blood cell
production
 Sinuses (vascular spaces) of the marrow are lined with
specialized endothelial cells, which prevent the premature
escape of immature cells into the peripheral blood.
 The basal lamina is incomplete, allowing mature cells to pass
through the wall of the sinuses.

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Bone marrow biopsy

 The clear space is an adipocyte

 the large cells with abundant pink cytoplasm and folded nuclei are
megakaryocytes;
 the small cells with opaque dark nuclei are late-stage erythroid
precursors;
 the cells with folded or bent nuclei are granulocytes.
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Bone Marrow Microenvironment
cont’d
 Stromal Cells compose the supportive tissues of the bone
marrow. Some of these cells produce hemopoietic growth
factors. Examples include:
◦ Adventitial (reticular) cells:
 Are modified fibroblasts that produce the reticulin
framework of the bone marrow
◦ Macrophages:
 Produce hemopoietic growth factors
 store iron for hemoglobin production, and
 carry out phagocytosis of debris
◦ Adipocytes: Store energy in the form of fat

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Regulation of Hemopoiesis
 Hemopoiesis is maintained in a steady state in which
production of mature cells equals cell loss
 Increased demands for cells as a consequence of
disease or physiologic change are met by increased
cell production.
 system subject to some form of feedback control
which could be exerted by humoral factors, e.g.,
 erythropoietin
 colony-stimulating factors
growth factors
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Feed back control e.g.
Increased destruction of red blood cells
(as in hemolytic anemia)

Low blood hemoglobin level

Tissue hypoxia

stimulation of increased erythropoietin

production by the kidneys

Increase in the rate at which committed

progenitor cells divide and differentiate

Hemoglobin level brought to normal 53

 Hemopoietic Growth Factors
Factor Function
Stem Cell Growth Stimulates pluripotent hematopoietic stem cells (hemocytoblasts)
Factor (Steel factor)

Interleukin-3 Stimulates pluripotent hematopoietic stem cells and progenitors of

(multi-CSF*) eosinophils, neutrophils, basophils, monocytes, and platelets
Granulocyte- Stimulates development of erythrocytes, platelets, granulocytes
Macrophage CSF (eosinophils, neutrophils, and basophiles,), and monocytes.
(GM-CSF)
Macrophage CSF Stimulates development of monocytes and macrophages
(M-CSF)
Granulocyte CSF Stimulates development of neutrophils
(G-CSF)
Interleukin-5 Stimulates development of eosinophils
Interleukin-7 Stimulates development of B lymphocytes
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*CSF=Colony stimulating factor
Hemopoietic Growth Factors and
site of action

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Summary of Hematopoiesis showing site of action of growth
factors
 GEMM=
Granulocyte/erythroid/mo
nocyte/megakaryocyte
precursor
 GM=
Granulocyte/monocyte
precursor
 GM-CSF= Granulocyte-
macrophage colony-
stimulating factor
 G-CSF= Granulocyte
colony stimulating factor
 IL-2= Interleukin 2
 IL-3= Interleukin 3
 IL-5= Interleukin 5
 IL-6= Interleukin 6
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 Maturation Characteristics
 Blood cells go through maturation stages in the bone marrow
and are released into the blood at maturity to perform their
function
 In any cell series, a progression of cells exists between the
most immature ‘blast’ cell and the mature cells
 Sometimes, it is difficult to know what stage is represented by
a particular cell
 The general rule is to identify the cell as the most mature
form.

57
Identification of cells
 Main features to identify cells on a Wright’s- stained smear are:
◦ Size of the cell
◦ Nuclear-cytoplasmic ratio
◦ Nuclear characteristics
 Chromatin pattern
 Nuclear shape
 Presence of nucleoli
◦ Cytoplasmic characteristics
 Color
 Granulation
 Vacuoles
 Shape

58
Identification of cells cont’d
Changes With maturation:
 Size of the cell decreases
 Nuclear:cytoplasmic ratio decreases from 4:1 or 3:1 to 2:1 or
1:1 in most cases
◦ Exceptions:
 erythrocytes and thrombocytes have no nuclei
 Small lymphocytes frequently retain the original ratio
 Nuclear characteristics

◦ Chromatin pattern becomes more coarse and dense

◦ Nuclear shape changes to many lobes or segments
(in Granulocytes)
◦ Nucleoli disappear
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Changes With maturation cont’d
 Cytoplasmic characteristics
◦ Color changes from deep blue color in the blast
stage to:
 lighter blue (e.g. lymphocytes)
 blue-gray (e.g. moncytes) or
 pink (e.g. RBC)
◦ Granulation: in the granulocytic series changes
from no granules in the blast stage to non-
specific granules then to specific granules

60
Changes With maturation
cont’d
 Cytoplasmic characteristics cont’d
◦ Vacuoles: vacuolation increases as the white cells
age (except for monocytes which frequently have
vacuoles throughout their life cycle)
◦ Shape: change of shape seen in the
megakaryocyte. It has more irregular outline
 In identifying of cells, examine more systematically by
assessing various maturational features

61
 Blast Cell Characteristics
 In blood cells developmental stages, the earliest morphologically
identifiable precursor is the blast cell
 Blast cell:
is a large cell
has round nucleus with fine chromatin and nucleoli,
has small amount of dark blue (Wright’s stain) cytoplasm,
is10-20 μm in diameter with high nuclear/cytoplasmic (N/C)
ratio
 Additional tests are needed to identify blasts in malignant
situations, such as the leukemias.
 The number of nucleoli varies depending on the cell type, as in the
following examples:

62
Blast Cell Characteristics cont’d
◦ Myeloblast: contains 1-5 nucleoli
◦ Lymphoblast: 1-2 nucleoli
◦ Monoblast: 1-2 nucleoli, but occasionally 3-4
◦ Erythroblast may have up to 2 that may stain darker
than other types of blast cells
◦ Megakaryoblast: has 1-5 nucleoli

63
Production Of Specific Cell Lines:
Erythrocyte Production
(Erythropoiesis)
 Erythropoiesis is the production of red cells
 Begins with the development of primitive erythrocytes in the
embryonic yolk sac
 Basic substances needed: are amino acids (proteins), iron, Vit
B12, Vit B6, folic acid and the trace minerals cobalt and nickel
 Regulated by erythropoietin, a glycoprotein primarily produced
by the kidneys in response to tissue hypoxia. (10-15%
production of erythropoietin occurs in the liver)
 Androgen and thyroid hormones can also stimulate
erythropoiesis

64
Erythropoiesis cont’d
 Erythroid precursors are derived from the CFU-GEMM
 The earliest progenitor committed exclusively to erythroid
lineage is the burst-forming unit–erythroid (BFU-E)
 This stage is followed by the colony-forming unit–erythroid
(CFU-E)
 The earliest recognizable RBC precursor is the
proerythroblast, which is characterized by fine nuclear
chromatin and intensely blue cytoplasm

65
Pronormoblast/Proerythroblast
(Rubriblast)
 Pronormoblast is the earliest morphologically recognizable red
cell precursor.
 Size: 20-25m in diameter.
 Nucleus:
large, round to oval
contains 0-2 light bluish, indistinct nucleoli
The chromatin forms a delicate network giving the nucleus
a reticular appearance.
 Cytoplasm:
there is a narrow (about 2m) rim of dark marine blue
cytoplasm
There may be a perinuclear halo
The N:C ratio is about 4:1

66
Basophilic
Normoblast/prorubricyte
 Size:16-18m in diameter.
 Nucleus:
Round or oval and smaller than in the previous stage
The chromatin forms delicate clumps so that its pattern
appears to be denser and coarser than that seen in the
pronormoblast.
No nucleoli are seen.
 Cytoplasm:
Slightly wider ring of deep blue cytoplasm than in the
pronormoblast
There may be a perinuclear halo
The N:C ratio is about 4:1

67
Polychromatophilic Normoblast/
Rubricyte
 Size:12-14m in diameter
 Nucleus:
smaller than in the previous cell
has a thick membrane
contains coarse chromatin masses
 Cytoplasm:
as the nucleus is shrinking the band of cytoplasm is
widening
It has a lilac (polychromatic) tint because of beginning of
hemoglobinization (blue layered with tinges of orange red
The N:C ratio varies from 2:1 to 4:1.

68
Orthochromatic Normoblast

 Size:10-12m in diameter.
 Nucleus:
small and central or eccentric with condensed homogeneous
structureless chromatin.
It is ultimately lost by extrusion.
 Cytoplasm:
a wide rim of pink cytoplasm surrounds the shrinking nucleus
 The entire cell is somewhat smaller than the
polychromatophilic normoblast
The N:C ratio varies from 1:2-1:3.

69
Reticulocyte
 Is a large somewhat basophilic anuclear cell formed after
the expulsion of the nucleus
 Remnants of RNA visualized as reticulum, filamentous
structure, in chains or as a single dotted structure when
stained with new methylene blue
◦ In Wright’s stain seen as large bluish-red cell,
Polychromatophilic macrocytes
 This network is responsible for the name of the cell and
consists of precipitated ribosomes.

70
Reticulocyte cont’d
 As the bone marrow reticulocyte matures the network
becomes smaller, finer, thinner, and finally within 3 days
disappears
 About 1% of reticulocytes enter the peripheral circulation
 Size: 8-10m in diameter
 Nucleus: the reticulocyte does not contain a nucleus.
 Cytoplasm: faintly basophilic (blue)

71
Mature erythrocyte

 Size:
7-8m in diameter
 Cytoplasm:

◦ Biconcave disc-shaped cell filled with hemoglobin

◦ orange-pink with a pale staining center occupying
one-third of the cell area (central pallor)

72
Erythropoiesis cont’d

73
Regulation of Erythropoiesis
 Erythropoietic activity is regulated by the hormone
erythropoietin which in turn is regulated by the level of tissue
oxygen
 Erythropoietin:
a heavily glycosylated hormone (40% carbohydrate) with a
polypeptide of 165 amino acids
Normally, 90% of the hormone is produced in the
peritubular (juxtaglomerular) complex of the kidneys
10% in the liver and elsewhere
There are no preformed stores of erythropoietin
the stimulus to the production of the hormone is the oxygen
tension in the tissues (including the kidneys)

74
Regulation cont’d
 Erythropoietin production increases when there is tissue
hypoxia due to:
◦ Low blood hemoglobin levels (e.g., anemia)
◦ Impaired oxygen release from hemoglobin for
some structural or metabolic defects (e.g., the
hemoglobinopathies)
◦ Poor blood flow as in severe circulatory defects
◦ Low atmospheric oxygen (e.g., high altitude)
 The produced erythropoietin stimulates erythropoiesis by
increasing the number of progenitor cells committed to
erythropoiesis

75
Regulation cont’d
 Erythropoietin accelerates nearly every stage of red cell
production:
 It increases the rate at which the committed stem cells divide
and differentiate
 It increases the rate of cell division
 It speeds up the incorporation of iron into the developing red
cells
 It shortens the time cell maturation, and
 It hastens the entry of reticulocytes into the peripheral
circulation

76
Regulation cont’d
 On the other hand, reduced erythropoietin activity is due to
increased oxygen supply to the tissues resulted from:
◦ Increased red cell mass (e.g., polycythemia)
◦ Ability of hemoglobin to release oxygen to the
tissues more readily than normal

77
Ineffective erythropoiesis/Intramedullary
hemolysis
 Erythropoiesis is not entirely efficient since 10-
15% of eryhtropoiesis in a normal bone marrow is
ineffective,
 the developing erythroblasts die within the marrow
without producing mature cells
 Together with their hemoglobin, they are ingested
by macrophages
 This process is substantially increased in a number
of anemias.

78
Megaloblastic Erythropoiesis
 Megaloblasts are pathologic cells that are not present in the
normal adult bone marrow
 their appearance is caused by a deficiency in vitamin B12 or
folic acid or both
 Deficiency of these vitamins leads to defective DNA
synthesis
 In megaloblastic erythropoiesis, the nucleus and cytoplasm
do not mature at the same rate
 Thus nuclear maturation lags behind cytoplasmic
hemoglobinization

79
Megaloblastic Erythropoiesis cont’d
 This nuclear lag appears to be caused by
interference with DNA synthesis while RNA
and protein synthesis continue at a normal rate
 The end stage of megaloblastic maturation is
the megalocyte which is abnormally large in
size (9-12m in diameter).

80
 Formation of white blood cells
(Leukopoiesis)
Granulopoiesis and Monocytopoiesis
◦ Neutrophils and monocytes arise form a common
committed progenitor
◦ The myeloblast is the earliest recognizable precursor in
the granulocytic series
◦ on division the myeloblast gives rise to promyelocyte
◦ The promyelocyte contain abundant dark “azurophilic”
primary granules that overlie both nucleus and
cytoplasm

81
Granulopoiesis cont’d
 with subsequent cell divisions these primary granules
become progressively diluted by the secondary, less
conspicuous “neutrophilic” granules that are characteristic
of the mature cells.
 This concomitant cell division and maturation sequence
continues form promyelocytes to early myelocytes, late
myelocytes, and then metamyelocytes
 As the metamyelocyte matures the nucleus becomes more
attenuated and the cell is then called a “band” or “stab”
form
 Subsequent segmentation of the nucleus gives rise to the
mature neutrophil or polymorphonuclear leucocyte.

82
Granulopoiesis cont’d
 The average interval from the initiation of granulopoiesis
to the entry of the mature neutrophil into the circulation is
10 to 13 days.
 The mature neutrophil remains in the circulation for only
about 10 to 14 hours before entering the tissue, where it
soon dies after performing its phagocytic function.

83
Myeloblast
 is the earliest recognizable precursor in the granulocytic series
 Size and shape:
 12-20 m in diameter
 round or oval in shape.
 Nucleus:
 large, oval or round, and eccentric.
 has a thin nuclear membrane
 has finely dispersed, granular, purplish, pale chromatin
with well-demarcated, pink, evenly distributed
parachromatin
 2-5 light blue-gray nucleoli surrounded by dense
chromatin are seen
84
Myeloblast cont’d
 Cytoplasm:

◦ is small in comparison to the nucleus

◦ High N:C ratio of 7:1
◦ stains basophilic (bluish) and shows a small
indistinct, paranuclear, lighter staining halo (golgi
apparatus)
◦ the cytoplasm lacks granules.

85
Promyelocyte
 largerthan the myeloblast
 Size and Shape:

◦ 15-20m in diameter and round or oval in shape.

 Nucleus:

◦ still large but is beginning to shrink

◦ round or oval, eccentric, possibly slightly indented,
and surrounded by a thin membrane
◦ 1-3 nucleoli may be faintly visible within the
finely of granular purplish pale chromatin,

86
Promyelocyte cont’d
 Cytoplasm:
 pale blue
 some what larger than in myeloblast, so the
nuclear/cytoplasmic ratio is 4:1 or 5:1
 the basophilia is not quite as intense as in
myeloblasts
 contain abundant dark “azurophilic” primary
granules that overlie both nucleus and cytoplasm
 these non-specific, peroxidase-containing
azurophilic granules are characteristic of the
promyelocyte stage of development

87
Promyelocyte
Myelocyte
 Isthe last stage capable of cell division
 Size and shape:
10-18m in diameter and round.
 Nucleus:
Condensed, oval, slightly indented, and eccentric
The chromatin is coarse
Nucleoli are absent.
 Cytoplasm:
Light pink and contains neutrophilic granules (brownish)
Granules that may cover the nucleus and are coarse in the
younger cells but become finer as the cell matures.
The N:C ratio is about 2:1 or 1.5:1

89
Metamyelocyte (Juvenile cell)
 Size and shape:
 10-15m in diameter and round.
 Nucleus:
 Eccentric, condensed, and indented or kidney-shaped
 The nuclear membrane is thick and heavy, and the
chromatin is concentrated into irregular thick and thin
areas.
 Cytoplasm:
 abundant and pale or pink
 contains both specific and non-specific (few) granules
that in the neutrophilic metamylocytes vary in size,
whereas the basophilic and eosinophilic granules are
large and equal in size.
 The NC ratio is 1:1
90
Metamyelocyte (bottom) and Band (top)
Band Granulocyte (Stab Cell)
 The juvenile cell or the band cell are the youngest granulocytes
normally found in the peripheral blood.
Size: 9-15m in diameter
Nucleus:
 elongated, curved and usually U shaped, but it may be twisted
 It is not segmented but may be slightly indented at one two
points
 The chromatin is continuous thick and coarse, and
parachromatin is scanty.
Cytoplasm:
 contains specific and a few non-specific granules
 is pink or colorless.
 The N:C ratio is 1:2

92
Segmented granulocyte
 Size: 10-12m in diameter.
 Nucleus:
eccentric with heavy, thick chromatin masses
It is divided into 2-5 lobes connected to each other by thin
bridges of chromatin membrane
The ratio of segmented to band forms is of clinical
significance and is normally about 10:1.
 Cytoplasm:
abundant and slightly eosinophilic (pinkish) or colorless,
and
contains specific granules
The neutrophilic granules are very fine in texture and do
not overlay the nucleus
The N:C is 1:2
93
Eosinophilic Granulocyte and Precursors
 Eosinophils mature in the same manner as neutrophils.
 The eosinophlic myeloblast is not recognizable as such.
 In the eosinophilic promyelocyte stained preparation the
granule are at first bluish and later mature into orange
granules
 The mature eosinophilic granules are

◦ larger than neutrophilic granules

◦ round or ovoid
◦ prominent in the eosinophilic myelocyte.

94
Mature Eosinophil
 Size and shape:
◦ 10-16m in diameter, slightly larger than a segmented
polymorphonuclear granulocyte.
 Nucleus:

◦ Eccentric
◦ usually bilobed
◦ rarely single- or tri-lobed and contains dense
chromatin masses.
◦ Eosinophils with more than two nuclear lobes are seen
in
 vitamin B12 and folic acid deficiency and
 in allergic disorders.

95
Eosinophil cont’d
 Cytoplasm:
 densely filled with orange-pink specific granules.
 The granules are
 uniform in size
 Large and individualized
 do not cover the nucleus
 Highly metabolic and contain histamine and other
substances

96
Basophilic Granulocyte and
Precursors
 The early maturation of the basophilic granulocyte is similar to
that of the neutrophlic granulocyte.
 Mature Basophil
 Size:

◦ Somewhat smaller than eosiniphils

◦ measuring 10-14m in diameter
 Nucleus:

◦ Indented giving rise to an S pattern.

◦ It is difficult to see the nucleus because it contains
less chromatin and is masked by the cytoplasmic
granules.
97
Basophils cont’d
 Cytoplasm:

◦ Pale blue to pale pink

◦ contains granules that often overlie the nucleus but
do not fill the cytoplasm as completely as the
eosinophilis granules do

98
Monocytes and their Precursors
Monoblast
 Since the monoblast cannot be differentiated from the
myeloblast on morphologic or histochemical criteria, one may
assume that the myeloblast can give rise to myeloid and
monocytic cells.
 Size: 15-20m in diameter.
 Nucleus:

◦ Round or oval and at times notched and indented

◦ The chromatin is delicate blue to purple stippling
with small regular, pink, pale or blue parachromatin
areas
◦ The nucleoli (3-5 in number) are pale blue, large and
round
99
Monoblast cont’d
 Cytoplasm:
 Relatively large in amount
 May contains a few azurophilic granules (rare)
 Stains pale blue or gray
 The cytoplasm filling the nucleus indentation is
lighter in color than the surrounding cytoplasm
 The surrounding cytoplasm may contain Auer
bodies.

100
Promonocyte
◦ Is the earliest monocytic cell recognizable as
belonging to the monocytic series
◦ is capable of mitotic division
◦ Its product, the mature monocyte, is only capable
of maturation into a macrophage
 Size:

◦ 12-20m in diameter.

101
Promonocyte cont’d
 Nucleus:

◦ Large
◦ ovoid to round, convoluted, grooved, and indented
◦ The chromatin forms a loose open network
containing a few larger clumps
◦ there may be two or more nucleoli.
 Cytoplasm:

◦ sparse, gray-blue, contains fine azurophilic

granules
 N:C ratio is about 3:1

102
 Monocyte
 Size:

◦ 12-20m in diameter.
 Nucleus:

◦ Eccentric or central
◦ Takes different shapes from brainy convolutions to
lobulated and S shaped (often lobulated)
◦ The chromatin network consists of fine, pale, loose,
linear threads producing small areas of thickening at
their junctions
◦ No nucleolus is seen
◦ The overall impression is that of a pale nucleus quite
variable in shape.
103
Monocyte cont’d
 Cytoplasm:

◦ Abundant, opaque, gray-blue with moderate

granules
◦ unevenly stained and may be vacuolated
 N:C ratio 1:1

104
Lymphopoiesis
 The precursor of the lymphocyte is believed to be the
primitive mulipotential stem cell that also gives rise to the
pluirpotenital myeloid stem cell for the granulocytic,
erythyroid, and megakaryocytic cell lines
 Lymphoid precursor cells travel to specific sites
 There, they differentiate into cells capable of either expressing
cell-mediated immune responses or secreting
immunoglobulins
 The influence for the former type of differentiation in humans
is the thymus gland;
◦ the resulting cells are defined as thymus-dependent
lymphocytes, or T cells.
105
Lymphopoiesis cont’d
 The site of the formation of lymphocytes with the potential
to differentiate into antibody-producing cells has not been
identified in humans, although it may be the tonsils or bone
marrow

 In chickens it is the bursa of Fabricius, and for this reason

these bursa-dependent lymphocytes are called B cells

B cells ultimately differentiate into morphologically distinct,

antibody-producing cells called plasma cells.

106
Lymphocytes and Precursors
Lymphoblast
 Size: 10-20m in diameter.
 Nucleus:
Central, round or oval
the chromatin has a stippled pattern
The nuclear membrane is distinct and one or two pink
nucleoli are present and are usually well outlined
 Cytoplasm:
Non-granular and sky blue
may have a deep blue border
It forms a thin perinuclear ring.
 N:C ratio 4:1

107
Prolymphocyte

 Size:

◦ 9-18m in diameter.
 Nucleus:

◦ Oval but slightly indented

◦ may show a faint nucleolus
◦ The chromatin is slightly condensed into a mosaic
pattern.
 Cytoplasm:

◦ Gray blue, mostly blue at the edges

◦ may show a few azurophilic granules and vacuoles
108
Lymphocytes
 There are two varieties
 the morphologic difference lies mainly in the amount of
cytoplasm
Small Lymphocyte
 Size:

◦ 7-18m in diameter.
 Nucleus:

◦ round or oval to kidney shaped

◦ occupies nine tenths of the cell diameter
◦ The chromatin is dense and clumped
◦ A poorly defined nucleolus may be seen.
109
Lymphocytes cont’d
 Cytoplasm:

◦ It is basophilic and forms a narrow rim around the

nucleus or at times a thin blue line only with few
azurophilic red granules
 N:C ratio is 4:1
 Distinguishing characteristics of a small lymphocyte:
◦ clumping of chromatin around the nuclear
membrane may help to distinguish this from a
nucleated red cell

110
Large Lymphocyte
 Size:

9-12m in diameter
 Nucleus:
the dense, oval, or slightly indented nucleus is centrally or
eccentricity located
Its chromatin is dense and clumped.
 Cytoplasm:
Abundant
gray to pale blue, unevenly stained, and streaked at times
A few azurophilic granules are contained in 30-60% of the
cells.
These are large granular lymphocytes (LGLs).

111
Large Lymphocyte cont’d

 N:C ratio is 4:1

 Distinguishing characteristics: Cytoplasm is more abundant
with tendency for azurophilic granules

112
Formation of platelets
(Thrombopoiesis)
 Platelets are produced in the bone marrow by fragmentation of
the cytoplasm of megakaryocytes
 The precursor of the megakaryocyte-the megakaryoblast-
arises by a process of differentiation for the hemopoietic stem
cell
 The megakaryoblast produces megakaryocytes, distinctive
large cell that are the source of circulating platelets.
 Megakaryocyte development takes place in a unique manner.

◦ The nuclear DNA of megakaryoblasts and early

megakaryocytes reduplicates without cell division,
a process known as endomitosis.

113
Thrombopoiesis cont’d
 As a result, a mature megakaryocytes has a polyploidy
nucleus, that is, multiple nuclei each containing a full
complement of DNA and originating from the same locust
within the cell.
 Mature megakaryocytes are 8 n to 36 n.
 The final stage of platelet production occurs when the mature
megakaryocyte sends cytoplasmic projections into the marrow
sinusoids and sheds platelets into the circulation.

114
Thrombopoiesis cont’d
 Ittakes approximately 5 days from a megakaryoblast to
become a mature megakaryocyte.
 Each megakaryocyte produces from 1000 to 8000 platelets.
 The platelet normally survives form 7 to 10 days in the
peripheral blood.

115
Morphology of the Platelets and their
Precursors
Megakaryoblast
 Size:
ranges from 10-30m in diameter.
The cell is smaller than its mature forms but larger than all
other blast cells.
 Nucleus:
the single, large, oval or indented nucleus has a loose
chromatin structure and a delicate nuclear membrane
Multi-lobulated nuclei also occur representing a polyploid
stage.
Several pale blue nucleoli are difficult to see
The parachromatin is pink.
116
Megakaryoblast cont’d
 Cytoplasm:

◦ the cytoplasm forms a scanty, bluish, patchy,

irregular ring around the nucleus
◦ The periphery shows cytoplasmic projections and
pseudopodia like structures.
◦ The immediate perinuclear zone is lighter than the
periphery.

117
Promegakaryocyte
 Size:
ranges from 20-50m in diameter.
It is larger than the megakaryoblast
in the process of maturation it reaches the size of the stage
III cell.
 Nucleus:
large, indented and poly-lobulated.
the chromatin appears to have coarse heavily stained
strands and may show clumping
The total number of nucleoli is decreased and they are
more difficult to see than in the blast cell.
The chromatin is thin and fine.

118
Promegakaryocyte cont’d
 Cytoplasm:

◦ intensely basophilic
◦ filled with increasing numbers of azurophilic
granules radiating from the golgi apparatus toward
the periphery sparing a thin peripheral ring that
remains blue in color.

119
Granular Megakaryocyte
 The majority of the megakaryocytes of a bone marrow aspirate
are in stage III which is characterized by progressive nuclear
condensation and indentation and the beginning of platelet
formation within the cytoplasm.
 Size:

◦ ranges from 30-100m in diameter

◦ is the largest cell found in the bone marrow.
 Cytoplasm:

◦ a large amount of polychromatic cytoplasm produces

blunt, smooth, pseudopodia-like projections that
contain aggregates of azurophilic granules surrounded
by pale halos
◦ These structures give rise to platelets at the periphery
of the megakaryocytes.
120
Platelets

Size:
◦ varies from 1-4m in diameter.
Nucleus:
◦ no nucleus is present.
◦ In Wright - Giemsa stained films, platelets appear
as small, bright azure, rounded or elongated bodies
with a delicately granular structure.

121
Review Questions/Summary
1. What is hemopoiesis and how is the process regulated?
2. What are the hemopoietic tissues during fetal life, in infancy,
in childhood and in adulthood?
3. What are the effects of the hormone erythropoietin on red cell
development and maturation.
4. Describe the microenvironment briefly.
5. Explain megaloblastic erythropoiesis.
6. Describe general Characteristic feature of cells during
maturation (nuclear , cytoplasmic, etc )
7. State the composition of blood.
8. State the main functions of blood.
9. List main characteristics of blood.
10. What is extramedulary hemopoiesis and when does it occur?

122