Hematology – Lecture

Module 2: Hematopoiesis │Canvas Notes │Medical Technology 2022

LESSON 3-1: GENERAL PRINCIPLES OF HEMATOPOIESIS • Bone marrow becomes the major site of hematopoiesis.
General Principles of Hematopoiesis Shortly after birth, the BM remains as the only tissue capable
Hematopoiesis (or Hemopoiesis) is the process of blood of blood cell production. When the BM is in distress or is not
cell production, including renewal, proliferation, differentiation, and functioning properly, secondary hematopoietic organs such as
maturation. It is a continuous and regulated process that results in the liver and spleen revert to their hematopoietic function
the formation, development, and specialization of all functional (extramedullary hematopoiesis)
blood cells that are released from the bone marrow. These mature • Erythropoietin, G-CSF, and GM-CSF (Growth Factors) reach
blood cells develop from a common hematopoietic stem cell. This detectable levels
stem cell is capable of both self-renewal and directed differentiation • Hemoglobins produced are HbA / HbA1, HbF, and HbA2
into the different cell lineages – ie. the hematopoietic stem cell can (minor adult hemoglobin)
proliferate and can give rise to any of the functional blood cell
SITES OF HEMATOPOIESIS IN THE DEVELOPING
lineages.
INDIVIDUAL
Hematopoiesis in humans is characterized by selective
distribution of embryonic stem cells in specific sites that are rapidly
changing during the course of development. In healthy adults,
hematopoiesis is primarily confined to the bone marrow. During fetal
development, blood cell development begins in the (1) yolk sac, that
later progresses to the AortaGonad-Mesonephros (AGM) region
(mesoblastic phase); (2) transfers to the fetal liver (hepatic phase);
and (3) terminally, resides in the bone marrow (medullary phase).

MESOBLASTIC PHASE
• Starts at the 19th day after fertilization
• Progenitor cells of mesenchymal origin relocate to the yolk
sac; give rise to Hematopoietic Stem Cells (HSCs)
• Erythroblasts (immature red blood cells) come from
mesodermal cells lining the yolk sac; remaining cells
surrounding the cavity develop into angioblasts and later on
form the blood vessels
• Yolk sac differs from other phases of hematopoiesis in that
yolk sac hematopoiesis occurs intravascularly (within the
blood vessels)
• Primitive erythroblasts are differentiated from later
erythroblasts in that primitive erythroblasts never lose their
nucleus. These erythroblasts are found in ‘blood islands’
surrounding a macrophage called Nurse cell.
• Primitive erythroblasts start to produce the following
hemoglobins: Portland hemoglobin, Gower 1
hemoglobin,and Gower 2 hemoglobin
• Some cells of mesodermal origin also transfer to the AGM
region to develop into HSCs for definitive hematopoiesis.
HEPATIC PHASE
• Begins at around 4-5 weeks after fertilization; Peaks at third
month of development
• The liver becomes the primary site of hematopoiesis
• Characterized by recognizable clusters of myeloid cells.
• Lymphoid cells begin to appear
• Megakaryopoiesis (development of platelet precursors, the
megakaryocytes) begins
• Sites of secondary hematopoiesis: Thymus begins to
produce T cells; Spleen and kidneys produce B cells
With detectable levels of HbF (fetal hemoglobin), HbA / HbA1
(major adult hemoglobin), and HbA2 (minor adult
hemoglobin.
• Activity remains until 1-2 weeks after birth
MEDULLARY PHASE
• Starts at the 5th month of development; cells of various stages
of maturation in all lineages are seen
• Mesenchymal cells transfer to the skeletal tissues and develop
into HSCs
• Myeloid to erythroid (M:E) ratio reaches 3:1 (adult M:E ratio)
at the 21st week
ORGANS INVOLVED IN HEMATOPOIESIS
1. Bone Marrow – primary site of hematopoiesis in an adult.
2. Liver – major site of hematopoiesis during the hepatic period.
3. Spleen – secondary site of hematopoiesis during the hepatic
period.
4. Thymus – secondary lymphoid organ; involved in the
maturation of T-cells
5. Lymph Nodes – secondary lymphoid organ; involved in
production of lymphocytes, filtration, and removal of old and
damaged cells.
6. Bursa equivalent organ – In humans, the Bursa-equivalent
organ is the bone marrow. In the Fabricius Bird, the bursa
is the site of maturation of B cells.
7. Mononuclear Phagocyte System
8. Kidneys – produces erythropoietin
9. Stomach – produces intrinsic factor
10. Yolk Sac – site of primitive erythropoiesis.
ADULT HEMATOPOIETIC TISSUE
Bone Marrow is the major site of hematopoiesis
Lymphoid development occurs in primary and secondary lymphoid
organs:
• Primary Lymphoid Organs: sites of maturation of
lymphocytes – Bone Marrow and Thymus
• Secondary Lymphoid Organs: sites of activation of
lymphocytes – Spleen, Lymph Nodes, Mucosa-associated
lymphoid tissue (MALT), Gut associated lymphoid tissue
(GALT).
BONE MARROW
• Composed of red marrow and yellow marrow; RED
MARROW is the hematopoietic tissue and the YELLOW
MARROW is composed of adipose.
• In Adults, red marrow is located in the sternum, skull,
vertebrae, scapulae, ribs, pelvic bones, and proximal ends
of long bones.
1|P age
Hematology – Lecture
Module 2: Hematopoiesis │Canvas Notes │Medical Technology 2022
• Ratio of Red Marrow to Yellow Marrow in the developing
individual:
o BEFORE BIRTH: 100% Red Marrow
o AT BIRTH: 90:10
o AT 19/20 Y/O: 60:40
o IN ADULTHOOD: 50:50
o AT 65 Y/O 40:60
• Yellow Marrow can revert to red marrow when there is
increased demand for hematopoiesis, such as in acute
blood loss and hemolysis.
• The Bone Marrow is the site of production and maturation
of myeloid cells – erythrocytes, megakaryocytes,
neutrophils, eosinophils, basophils, and monocytes.
• The bone marrow produces the lymphocytes, The B cells
mature in the bone marrow. The T cells, however, mature
in the Thymus. The lymphocytes are activated in
secondary lymphoid organs.
LIVER
• Plays a significant role in hematopoiesis during fetal life
(hepatic phase)
• Responsible for synthesis of most proteins and vitamins
that play a role in regulating hemostasis
• Responsible for detoxification of blood
• Site of protein synthesis and degradation
• Kupffer cells lining the canaliculi remove senescent and
damaged red blood cells from circulation as they pass
through the liver.
SPLEEN
• Largest lymphoid organ in the body; secondary site of
hematopoiesis during hepatic phase.
Functions of the Spleen:
o Culling: removal of senescent (old) red blood
cells from blood circulation by phagocytosis.
o Pitting: removal of inclusion bodies from the
surface of red blood cells (ex. Pappenheimer
bodies – accumulated iron; Howell – Jolly bodies
– DNA remnants; Heinz bodies – globin
remnants.)
o Immune Defense: It is a secondary lymphoid
organ, serving as a site of activation of
lymphocytes (B and T cells)
o Storage of Platelets: the spleen sequesters 1/3
of platelets produced to serve as reservoir.
THYMUS
• Primary lymphoid organ; secondary site of hematopoiesis
during hepatic phase.
• Site of maturation of T cells.
LYMPH NODES
• Secondary lymphoid organ
• Site of activation of lymphocytes
• Filters debris, particulate matter, and bacteria from the
lymph.
• Serves as site of proliferation of lymphocytes.
MONONUCLEAR PHAGOCYTE SYSTEM
• Composed of the monocytes and macrophages
FUNCTION:
o Phagocytosis: removal of debris, particulate
matter, and foreign cells from the blood
(monocytes) and tissues (macrophages)
o Antigen presentation: antigens from digested
foreign cells (bacteria) are presented to T cells
for activation of the adaptive immune system.
o Mitogen: substances that promote mitosis
secretion
o Secretion of hematopoietic growth factors
(substances that influence the maturation and
differentiation of blood cells.
KIDNEYS
• Responsible for production of erythropoietin (growth factor
that drives maturation of RBC precursors) in response to
hypoxia. Erythropoietin acts on erythroblasts in the bone
marrow to stimulate proliferation and maturation, for
eventual release into the circulation.
STOMACH
• Produces intrinsic factor. Intrinsic Factor is necessary for
absorption of Vit. B12 in the intestines. Deficiency of IF
leads to deficiency in Vit. B12 and would lead to pernicious
anemia (a type of Megaloblastic anemia)
STEM CELL THEORY
Stem cells are characterized by its ability for/ to:
1. Self-renewal;
2. Give rise to differentiated progeny (i.e. hematopoietic stem
cell can differentiate into a common myeloid stem cell or
common lymphoid stem cell to later on give rise to mature
and functional blood cells);
3. Reconstitute the hematopoietic system in a lethally
irradiated individual.
Normal cell development depends the interaction of:
1. Pluripotent stem cell
2. Microenvironment
3. Hematopoietic Growth Factors
A pluripotent hematopoietic stem cell can be stimulated to
undergo one of three possible fates: self-renewal, differentiation, or
apoptosis. When the stem cell divides, it gives rise to two identical
daughter cells. The daughter cells may likewise be stimulated to
undergo any of the three outcomes. A stem cell can also be
stimulated for differentiation - eg. An HSC can differentiate into a
common myeloid stem cell (common myeloid progenitor) or a
common lymphoid stem cell (common lymphoid progenitor). The
common myeloid stem cell may differentiate into committed
(lineage-specific) precursor cells such as a Proerythroblast to
eventually give rise to mature erythrocytes or Megakaryoblast to
give rise to platelets. Differentiation of stem cells and maturation of
precursor cells occurs under the influence of hematopoietic growth
factors and under optimal conditions of the microenvironment.
Cytokines and Growth Factors: a group of glycoproteins that
regulate the proliferation, differentiation, and maturation of
hematopoietic precursor cells. Cytokines can either promote or
inhibit proliferation, differentiation, and maturation of blood cells.
Cytokines may also inhibit apoptosis (programmed cell death),
allowing cells to proliferate. Cytokines may be Colony Stimulating
Factors (CSF), early-acting multilineage growth factors or
interleukins
2|P age
Hematology – Lecture
Module 2: Hematopoiesis │Canvas Notes │Medical Technology 2022
Maturation Series of Erythroid Cells:

Normoblastic Erythroblastic Rubriblastic

Nomenclature Nomenclature Nomenclature
Pronormoblast Proerythroblast Rubriblast
Basophilic Basophilic Prorubricyte
Normoblast Erythroblast
Polychromatic Polychromatic Rubricyte
(Polychromatophili (Polychromatophili
c) Normoblast c) Erythroblast
Orthochromic Orthochromic Metarubricyte
Normoblast Erythroblast
Polychromatic Polychromatic Polychromatic
(Polychromatophili (Polychromatophili (Polychromatophili
c) Erythrocyte** c) Erythrocyte** c) Erythrocyte*
Erythrocyte Erythrocyte Erythrocyte
*This stage is also called as the RETICULOCYTE STAGE

Key features of the different stages of development of

RBCs:

The diagram above shows the derivation of hematopoietic stem

Pronormoblast/ Rubriblast
cells and the sites of action of cytokines. From Rodak’s Hematology:
• Diameter: 12-20 um
Clinical Principles and Applications (Keohane, EM, Smith, LJ, and
• N:C Ratio: 8:1
Walenga, JM) 2016
• Nucleus with 1-2 nucleoli
General Changes undergone by hematopoietic cells as they • Basophilic cytoplasm
mature: • Heme production starts in the mitochondria and globin
1. As the cells mature, they decrease in size. synthesis in the ribosomes begins
2. As the cells mature, cytoplasmic staining becomes less • May undergo mitosis
basophilic.
3. As the cells mature, Nucleus-to-Cytoplasm (N:C) ratio Basophilic Normoblast/ Prorubricyte
decreases. • Diameter: 10-15 um
4. As the cells mature, nuclear chromatin becomes denser. • N:C Ratio: 6:1
5. As the cells mature, nucleoli start to disappear. • Parachromatin area of the nucleus becomes larger;
staining is purple-red
*These are the general changes. Some lineages might have some • Nucleus has 0-1 nucleolus
exceptions to these rules* • Basophilic cytoplasm (darker than cytoplasm of PN)
LESSON 3-2: ERYTHROPOIESIS • Hemoglobin synthesis starts
• May undergo mitosis

ERYTHROPOIESIS Polychromatic Normoblast/ Rubricyte

Erythropoiesis refers to the production and development • Diameter: 10-12 um
of red blood cells. This process is largely influenced by • N:C Ratio: 4:1
erythropoietin (EPO). Erythropoietin acts on erythroblasts • Nuclear chromatin strands denser
(normoblasts; red blood cell precursors) in the bone marrow, • No visible nucleoli
promoting proliferation and maturation. • Cytoplasm: combination of blue and pink (murky gray-
bblue on Wright-strained smear
From the CFU-GEMM (Colony-Forming Unit – • Last Stage of Mitosis
Granulocyte, Erythrocyte, Monocyte, Megakaryocyte; Common
Myeloid Stem Cell), the earliest identifiable colony of RBCs arises, Orthochromic Normoblast/ Metarubricyte
the BFU-E (Burst Forming Unit – Erythroid). The BFU-E gives rise • Diameter: 8-10 um
to CFU-E (Colony-Forming Unit – Erythroid) under the influence of • Pyknotic Nucleus (condensed) without nucleoli
IL-3, GM-CSF, Thrombopoietin (TPO), and KIT Ligand. The • N:C Ratio: 1:2
CFUE, under the influence of EPO differentiates into the erythroid • Cytoplasm stains pink due to predominance of Hgb
precursor Pronormoblast. Further differentiation and maturation is • Last stage with a nucleus
also under the influence of EPO.

Under normal conditions, the kidneys produce a small

amount of erythropoietin. But under conditions of stress decreasing
oxygen supply (hypoxia) to the tissues, the kidneys respond to the
stimulus by increasing secretion of erythropoietin. This increased
EPO output likewise increases RBC production and release from
the bone marrow.

3|P age
Hematology – Lecture
Module 2: Hematopoiesis │Canvas Notes │Medical Technology 2022
Granulopoiesis
The granulocytes – the neutrophil, the eosinophil, and the
basophil – have similar stages of development. Although the stages
of development are similar, that is not to say that they develop from
the same precursor - ie. the myeloblast stage is a stage that is
common to all three cell lines, but the myeloblast is already
committed to a single cell line. But because the stages of
development are the same, development of these three cell lines
are often discussed as a group.

Growth Factors involved in differentiation and maturation

are GM-CSF and G-CSF.

Stages of Development of Granulocytes

Reticulocyte
• Diameter: 8-10 um
• Anucleate
• Cytoplasm stains pink
• Reticulum (cytoplasmic
RNA remnants) may be
stained with supravital
stains (e.g., New
Methylene Blue) but not
with ordinary stains
• Present in BM (about 2
days) but later released into the peripheral blood
• Last stage where hemoglobin synthesis occurs
• Circulates as reticulocyte for 1 day before it becomes a It is important to note, again that each of the three cell lines do not
mature erythrocyte. develop from the same Myeloblast. The Neutrophil develops from
its own precursor, the Neutrophilic Myeloblast. Although for practical
Erythrocyte
purposes, because all three myeloblasts look alike, they are simply
• Diameter: 7-8 um
referred to as ‘Myeloblast.’
• Biconcave shapes
• Has no mitochondria Summary of Morphological Changes in Granulocyte
• On a Wright-stained Development (Romanowsky-Stained)
smear, appears as
salmon-pink or red-
staining cell with a central
pallor area
• Circulated in the blood for
120 days before being removed by splenic or liver
macrophages

LESSON 4: LEUKOPOIESIS

LEUKOPOIESIS
Leukopoiesis refers to the production, development, and
maturation of white blood cells. Granulocytes and Monocytes
develop from the common myeloid stem cell (CFU-GEMM) while the
lymphocytes develop from the common lymphoid stem cell.
Leukopoiesis is subdivided into three categories:

1. Granulopoiesis: development and maturation of

Neutrophils, Eosinophils, and Basophils;
2. Monopoiesis: development and maturation of
Monocytes and Macrophages: and
3. Lymphopoiesis: development and maturation of
Lymphocytes.

4|P age
Hematology – Lecture
Module 2: Hematopoiesis │Canvas Notes │Medical Technology 2022
• Charcot-Leyden Crystal Protein (Eosinophil only)

MYELOBLAST CONTENTS OF NEUTROPHIL SECONDAY GRANULES

- Earliest stage recognizable under the light microscope. • β2-microglobulin
• Collagenase
There are three types of Myeloblast:
• Gelatinase
• Type I Myeloblast: no visble granules when viewed • Lactoferrin
under the light microscope • Neutrophil gelatinase-associated lipocalin (NGAL)
• Type II and III Myeloblast: granular when observed • Transcobalamin
under the light microscope.
CONTENTS OF NEUTROPHOL TERTIARY GRANULES
PROMYELOCYTE • Gelatinase
- Stage of synthesis og primary granules (aka non-specific • Collagenase
granules; azurophilic granules) • Lysozyme
• Acetyltransferase
MYELOCYTE • β2-microglobulin
- Last stage of capable of mitosis
- Stage of synthesis of Secondary (specific) granules – CONTENTS OF EOSINOPHIL SECONDARY GRANULES
these granules are lineage-specific • Major Basic Protein
• Eosinophil cationic protein
METAMYELOCYTE
• Eosinophil-derived neurotoxin
- Stage which is most abundant in normal bone marrow;
• Eosinophil peroxidase
stage of development of tertiary granules (for neutrophils
only) • Lysozyme
- Nucleus if slightly indented. • Catalase
• β-glucoronidase
BAND • Cathepsin D
- Stage of granulocyte development that first appears in • IL-2, IL-4, IL-5, IL-6
peripheral blood; granules are the same granules of • GM-CSF
mature granulocytes; has a deeply indented nucles (C-,S-
,Z, shaped) that may fold on itself. CONTENTS OF BASOPHIL SECONDARY GRANULES
• Histamine
MATURE (SEGMENTED) NEUTROPHIL • Platelet-activating factor
- Has fine lilac to purple granules in the cytoplasm; nucleus • Leukotriene C4
has 2-5 segments (thus, also known as
• IL-4 and IL-13
Polymorphonuclear neutrophil/ PMN)
• Vascular endothelial growth factors A and B
MATURE EOSINOPHIL • Chondroitin sulfate (eg. Heparan sulfate)
- Has coarse orange or pink granules in the cytoplasm that
MONOPOIESIS
do not oberlap with the nucleus; nucleus is often bi-lobed.
Development and maturation of monocytes. Monocytes are
MATURE BASOPHIL precursors of macrophages. The major cytokine responsible for
- Has coarse bluish-black granules obscruing the view of monopoiesis is the M-CSF.
the nucleus; nucleus if more often not visible when viewed
Summary off Morphological Changes in Monocyte
under the light microscope.
Development

12-20um in diameter; with basophilic

cytoplasm; high N:C ratio (3:1 to 4:1); with 1-2
Monoblast
nucleoli; similar in appearance to the
Myeloblast
14-18um in diameter; with blue-gray
cytoplasm; N:C ratio is 2:1 to 3:1; with 0-1
Promonocyte
nucleolus; last stage capable of mitosis;
confined to the bone marrow
15-20um in diameter; with blue-gray
cytoplasm (often described as having ground
glass appearance); with a kidney bean-shaped
Monocyte nucleus (described as having brain-like
convolutions); it is the only stage found in the
CONTENTS OF PRIMARY (AZUROPHILIC) GRANULES peripheral blood; it is the largest cell in
• Myeloperoxidase circulation
• Acid-β-glycerophosphatase 15-85 um in diameter; with a round to reniform
Macrophage
• Cathepsins nucleus; found in peripheral tissues
• Defensins
• Elastase
• Proteinase-3

5|P age
Hematology – Lecture
Module 2: Hematopoiesis │Canvas Notes │Medical Technology 2022
LYMPHOPOIESIS
Production, Development, and Maturation of Lymphocytes.

Lymphocytes are divided into three major groups; 1. T Cells, 2. B

Cells, and 3. Natural Killer (NK Cells)

Summary of Morphological Changes Occurring in the

Lymphocytes Development

In lymphocyte development, lymphoblasts are produced in

the bone marrow. The development of B cells and T cells differ in
their location. For B cells, lymphoblasts differentiate into pro-B cells,
which later become pro-B cells and immature B cells within the Bone
Marrow. Immature B cells leave the Bone Marrow and relocate into
secondary lymphoid organs where they may be activated. Activated
B cells may undergo mitosis, become memory B cells, or become
antibody-producing plasma cells.

T cell development on the other hand, occurs in the

Thymus. Lymphoblasts in the Bone Marrow relocate to the Thymus
where they differentiate into pro-T, pre-T, and later on immature T
cells. These immature T cells then transfer to secondary lymphoid
organs for activation. Activated T cells may either become effector
T cells or memory T cells. Activation of T cells causes what are seen
in peripheral blood as medium or large lymphocytes.

6|P age