Molecular biology

1. CHAPTER 2
Composition, Formation and
Function of Blood

2. Learning Objectives
At the end of this chapter, the student shall be
able to:
Explain the composition of blood.
Describe the morphology and functions of the
formed elements of blood.
Discuss the functions of plasma.

3. Cont…
Define hemopoiesis and explain the process of
blood cell origin and development.
 Indicate the sites of hemopoiesis in infancy,
childhood and adulthood.
Name the cells in the development order that
will mature into erythrocytes, thrombocytes
and the five leukocytes.

4. Cont…
Explain the regulatory mechanisms in
hemopoiesis
Describe the morphology of the red blood cell,
white blood cell, and platelet precursors
 Differentiate between intramedulary and
extramedulary hemopoiesis.

5. Cont…
• 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 to erythroid ratio.

6. 2.1 Composition of Blood
Blood is :
• the only fluid tissue.
• constitutes 6-8% of the total body weight.
• consists of cells suspended in a fluid called
plasma.
• about 45% cells; 55% plasma.

7. Cont…
Major Components of whole blood

8. Composition cont’d
1. Plasma
 is a straw-colored, sticky fluid.
 part of the extracellular fluid
 a complex solution of proteins, salts and numerous
metabolic substances
 acts as a transport medium carrying its constituents to
specialized organs of the body.

9. Cont…
Components are :
• Water = 92%
• Protein = 7%
• The remaining 1% is
• Dissolved organic molecules (glucose, amino
acids, lipids, and vitamins)
• Ions (Na+, K+, Cl-, H+, Ca++, HCO3
-) … Na+ and
Cl- are the most abundant electrolytes (ions) and
trace elements

10. Cont…
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%

11. Cont…
• Serum vs Plasma –
• Both are fluid portions of blood
• Serum is from clotted blood, has no clotting
factors (used up in the clotting process)
• Plasma is from anticoagulated blood, has
clotting factors, makes up about 45-60% of
blood‟s volume .

12. 2.Formed Elements
• The three main blood cells/formed elements
are:
 red blood cells (erythrocytes)
 white blood cells (leucocytes)
Platlets (thrombocytes )

13. Cont…

14. 2.1 Erythrocytes (Red Blood Cells)
• Are the most numerous cells in the blood.
• Red in color because of hemoglobin, which binds
and transport oxygen and carbon dioxide.
• about 45% of total blood volume in an adult, giving
blood its red colour.
• The normal RBC count is approximately 4.5 to 6
million cells per microliter, the exact number varying
with age, gender, and state of health.

15. Cont…
• Anucleate, no centrioles, no organelles
End result - no cell division
No mitochondria means they generate ATP
anaerobically
• Prevents consumption of O2 being transported
• a large proportion of their cytoplasm consists of the
iron containing oxygen transport molecule
hemoglobin.
• shaped like biconcave disks approximately 7 to 8
µm in diameter with a thickness of 1.7-2.4 µm.

16. Cont…
• 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.

17. Cont…
• 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.

18. Cont…
 Note that the size of the erythrocytes is about the same
as the nucleus of the small resting lymphocyte.

19. Cont…
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.

20. 2.2 Leukocytes (White Blood Cells)

21. Cont…
• 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/uL (4.0–
10.0 x 103 /uL)
• Leukocytes are usually divided into:
 Granulocytes, which have specific granules, and
 Agranulocytes, which lack specific granules.

22. Cont…
• Granulocytes/ Polymorphonuclear leukocytes are
divided into:
 Neutrophils
 Eosinophil
 Basophils
• Agranulocytes/mononuclear leukocytes are divided
into:
 Lymphocytes and Monocytes.
• Although they are called white blood cells,
leukocytes predominantly function in tissues.

23. 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 10-12um 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.

24. Cont…
• Normal range: 2.0-7.5 x 103 /ul.
• Increased in acute bacterial infections.

25. Eosinophils
• Have the same size as neutrophils or may be a bit
larger (12-14um).
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.

26. Cont…
• Normal range: 40-400/ul.
• Increase in their number (eosinophilia) is
associated with allergic reactions and
helminthiasis.

27. Basophils
• Size: 10-12um 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.
• They destroy microorganisms by releasing important
substances from their granules such as:
– heparin (inhibitors of blood clotting)
– histamine, serotonin, cytokine
– Proteases and myeloperoxidase

28. Cont…
• involved in immediate hypersensitivity
reactions related to immunoglobulin class E
(IgE)
• Normal range: 20-200/ul.
• Basophilia is rare except in cases of chronic
myeloid leukemia.

29. 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/ul.
• The lymphocyte number is higher in children
and also increases with viral infections.

30. Cont…
• There are two varieties:
1. Small Lymphocytes
 Their size ranges from 7-10µm in diameter.
 have round, deep-purple staining nucleus which
occupies most of the cell.
 There is only a rim of pale blue staining
cytoplasm.
 They are the predominant forms found in the
blood.

31. Cont…
2. Large Lymphocyte
• A small number of lymphocytes in the blood
• Slightly larger than resting lymphocytes
• This appearance generally corresponds to
natural killer (NK) cells.
• Size: 12-14 um in diameter.

32. Cont…
• 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.
 small lymphocye is shown for
comparison

33. Monocytes
• Are the largest white cells measuring 14-18um
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).

34. Cont…
• Cytoplasm is :
o abundant staining light gray to light blue
o finely granular.
• Nucleus has very finely granular chromatin
and is often folded, bean shaped, oval, or
irregular.

35. Cont…
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/ul.
Monocytosis is seen in bacterial infections
(e.g., tuberculosis) and protozoan infections.

36. Cont…
note the vacoules in the monos

37. Cont…

38. 2.3 Platelets (Thrombocytes)
• are small, non nucleated (anucleated), round/oval
cells/cell fragments.
• Their size ranges 1-4um 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.

39. Cont…
• Platelets have a life span of approximately 10
days.
• Senescent platelets are removed by the spleen
• Normal range: 150-400 x 103 /ul.
note: Platelets are shown by the arrow; the big cell in the
film is a basophil.

40. Characteristics of Blood
1.Temperature
Roughly 38°C (100.4 °F) ,slightly higher than “normal”
body temperature.
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

41. Cont…
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).

42. Cont…
 Hypovolemic = below normal
Normovolemic = normal.
Hypervolemic = above normal
Abnormally high BV can place severe stress on
the heart(pushing extra fluid through circulatory
system stresses heart).

43. Function of Blood
1.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
2. Regulation
• pH
• Temperature
• Osmotic pressure (influence water and ion content of
cells)

44. Cont…
3. Protection
• From bleeding (by the clotting mechanism)
• Immunity (phagocytes, lymphocytes,
antibodies, complement proteins, etc)

45. Function of plasma components
• Water – is a medium for carrying other
constituents i.e it is used as a solvent
• Inorganic substances – include Na- , K+ , Cl- ,
Ca2+ , Respiratory gase s, etc.
• Organic Substances
A. Glucose, Amino acid and fatty acid –
 Use plasma as means of transportation to their
site of utilization.
They are used as building materials for larger
molecules and used as energy sources, etc

46. Cont…
B. Proteins
Used to bind other plasma constituents such as lipids,
hormones, vitamins, metals etc, some are used as
enzymes or enzyme precursors.
• Albumin
helps to keep water from diffusing out of the
bloodstream into the extracellular matrix of tissues.
 Contributors to the osmotic pressure of plasma.

47. Cont…
• Globulin:
α-is associated with the transport of Bilirubin,
lipid and steroids.
- act as a substrate for formation of other
substances.
 β- for transport of Fe and Cu in plasma.
- for antibody production.

48. Cont…
Fibrinogen is the precursor for fibrin and is used
in hemostasis.
involved in a series of chemical reactions that
achieves blood clotting.
It forms large, and insoluble strands of fibrin

49. 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

50. 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.

51. 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

52. Hematopoiesis 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.

53. Hematopoiesis cont’d

54. Hematopoiesis cont’d

55. Stages in hemopoietic cell
development

56. 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.

57. 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.

58. Site of hemopoiesis

59. Stages/Phases of hemopoiesis in the
embryo and fetus
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.

60. 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.

61. Medullary hemopoiesis
• Blood cell production within the bone marrow
(medulla)

62. 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)

63. Extramedulary hemopoiesis …

64. 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.
an extracellular matrix of fibronectin, collagen and
laminin combines with these cells to provide a setting
in which stem cells can grow and divide.

65. …

66. 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.

67. 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.

68. Bone Marrow Microenvironment
cont‟d

69. • 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.
Bone Marrow Microenvironment
cont‟d

70. Bone Marrow Microenvironment
cont‟d

71. 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

72. 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

73. Hemopoietic Growth Factors
Factor Function
Stem Cell Growth
Factor (Steel factor)
Stimulates pluripotent hematopoietic stem cells (hemocytoblasts)
Interleukin-3
(multi-CSF*)
Stimulates pluripotent hematopoietic stem cells and progenitors of
eosinophils, neutrophils, basophils, monocytes, and platelets
Granulocyte-
Macrophage CSF
(GM-CSF)
Stimulates development of erythrocytes, platelets, granulocytes
(eosinophils, neutrophils, and basophiles,), and monocytes.
Macrophage CSF
(M-CSF)
Stimulates development of monocytes and macrophages
Granulocyte CSF
(G-CSF)
Stimulates development of neutrophils
Interleukin-5 Stimulates development of eosinophils
Interleukin-7 Stimulates development of B lymphocytes
*CSF=Colony stimulating factor

74. Hemopoietic Growth Factors and site of
action

75. Summary of Hematopoiesis showing site of
action of growth factors
• GEMM=
Granulocyte/erythroid/mon
ocyte/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

76. 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.

77. 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

78. …
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
•

79. …
• 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
– 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

80. 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:

81. …
– 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

82. 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.

83. 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 (Pronormoblast), which is
characterized by fine nuclear chromatin and intensely
blue cytoplasm.

84. 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

85. 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
– corn flower blue with indistinct areas of clearing.
– The N:C ratio is about 4:1

86. 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.

87. 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.

88. 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.

89. Reticulocyte cont’d
Reticulocytes after counter staining with
Romanowsky dyes (left pannel) and supravital
dyes (right)

90. 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).

91. 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)

92. Erythropoiesis cont’d

94. 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).

95. 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.

96. 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.

97. Regulation cont’d
• On the other hand,reduced erythropoitin 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.

98. 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.

99. • 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.
Megaloblastic Erythropoiesis

100. Megaloblastic Erythropoiesis cont’d
• Thus nuclear maturation lags behind cytoplasmic
hemoglobinization.
• 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).

101. 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
– with subsequent cell divisions these primary granules
become progressively diluted by the secondary, less
conspicuous “neutrophilic” granules that are characteristic
of the mature cells.

102. Granulopoiesis cont’d
• 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.

103. 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.

104. Granulocyte maturation
• Myeloblast
• Promyelocyte
• Myelocyte
(B,N,E)
• Metamyelocyte
(B,N,E)
• Band (B,N,E)
• Segmented
(B,N,E)
Immature WBCs, granulocytes Mature WBCs

105. 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

106. 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.

107. Promyelocyte
• larger than 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.

108. 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.

109. Myelocyte
• Is the 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

110. 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

111. Band Granulocyte (Stab Cell)
• 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

112. 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

113. 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.

114. 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.

115. 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

116. 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.

117. 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.

118. 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

119. 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.

120. 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.

121. 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

122. 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.

123. Monocyte cont’d
• Cytoplasm:
– Abundant, opaque, gray-blue with moderate
granules
– unevenly stained and may be vacuolated
• N:C ratio 1:1

124. 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;

125. Lymphopoiesis cont’d
• the resulting cells are defined as thymus-dependent
lymphocytes, or T cells.
• 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.

126. 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

127. …
• Cytoplasm:
– Non-granular and sky blue
– may have a deep blue border
– It forms a thin perinuclear ring.
• N:C ratio 4:1
• Distinguishing feature for a lymphoblast: Nucleoli is
surrounded by a dark rim of chromatin

128. 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.

129. 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.

130. 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

131. 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).
– Distinguishing characteristics: Cytoplasm is more
abundant with tendency for azurophilic granules.

132. 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.

133. Thrombopoiesis cont’d
• 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.
• 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.

134. Thrombopoiesis cont’d
• The final stage of platelet production occurs when the
mature megakaryocyte sends cytoplasmic projections
into the marrow sinusoids and sheds platelets into the
circulation.
• It takes approximately 5 days from a megakaryoblast
to become a mature megakaryocyte.
• Each megakaryocyte produces from 1000 to 8000
platelets.
• The platelet normally survives for 7 to 10 days in the
peripheral blood.

135. 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.

136. 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.

137. 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.

138. 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.

139. 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.

140. 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.

141. Summary
• Blood cell production, maturation, and death occur in
organs of the reticuloendothelial system (RES).
• RES includes bone marrow, spleen, liver, thymus,
lymph nodes.
• RES functions in hematopoiesis, phagocytosis, and
immune defense.

142. …
• Intrauterine hematopoiesis includes three phases:
a. Mesoblastic (yolk sac) phase begins at -19 days
gestation. The yolk sac is located outside the
developing embryo. The first cell to be produced is a
primitive nucleated erythroblast. This cell produces
embryonic hemoglobins.
b. Hepatic (liver) phase begins at 6 weeks gestation
with production of mainly red blood cells, but also
granulocytes, monocytes, and megakaryocytes

143. …
C. Myeloid/medullary phase begins around the fifth
month of gestation, with the bone marrow producing
mainly granulocytes.
• The M:E (myeloid:erythroid) ratio approaches the
adult level of 3: I.

144. …

145. Review Questions
1. State the composition of blood.
2. State the main functions of blood.
3. List main characteristics of blood.
4. What is hemopoiesis and how is the process regulated?
5. What are the hemopoietic tissues during fetal life, in
infancy, in childhood and in adulthood ?
6. What are the effects of the hormone erythropoietin on
red cell development and maturation.
7. What is extramedulary hemopoiesis and when does it
occur?