Hematology

HEMATOPOIESIS
-

blood cell production

encompasses the overall interaction of:
a. cellular proliferation
b. differentiation
c. morphogenesis
d. functional maturation
e. death

Periods of Hematopoietic System Development

Mesoblastic Period

begins during embryonic development

first detected at 19 to 20 days of gestation
primarily responsible for red cell production
Erythroblasts - immature red cells produced in the yolk sac
- are found after the third month of gestation

Primitive Erythroblasts - are large and cannot extrude their nucleus

- produce globin chains epsilon & zeta
- responsible for embryonic hemoglobin
3 Embryonic Forms of Hemoglobin
1. Gower I
2. Gower II
3. Portland

Hepatic Period

Fetal Liver - primarily an erythroid organ

- produces cells containing fetal hemoglobin (2 and 2 )
Liver - primary site of blood cell production until the 6th month
- may continue to produce cells until the 1st or 2nd week but in
smaller degree

Other Organs Active in Blood Production

1. Spleen

2. Thymus
3. Lymph Nodes
Extramedullary Hematopoiesis - blood cell production outside of the
bone
marrow

Myeloid Period

during the first few weeks of life, a balance between the developing
bone marrow space and the developing infants need for blood cells
hematopoietic capability of liver and spleen remain available
during the fourth year of life, rate of bone marrow growth exceeds
the need for blood
active marrow sites are replaced with areas of fatty reserve
fat first develops in shafts of bones until the age of 18
Only Active Hematopoietic Sites After 18

1. Pelvis
2. Vertebrae
3. Ribs

4. Sternum
5. Skull
6. Proximal Extremities of the Long Bones
Bone Marrow - main site of blood production because bone cavities begin
to form on the 5th month of gestation
- becomes the only normal site of blood cell production
during the first 3 weeks of postpartum and remains so
throughout life

Stem Cell Theories and Cell Production

Monophyletic Theory / Unitarian Theory

replenishment of blood cells is dependent on the presence of

undifferentiated hematopoietic cells (stem cells)
- studies demonstrated that all hematopoietic cells originate from

common cell termed : pluripotential stem cell

Stem Cell - products of tissue development
- produce all other cells
Stem Cell Pool - the population of pluripotential stem cells
- provides adequate number of stem cells for
differentiation into various cell lines
THSC - totipotential hematopoietic stem cell
- gives rise to both myeloid and lymphoid stem cell
Committed Stem Cell - differentiation of stem cells continue along a
specific pathway wherein they lose their potential
to develop as ay other cell
Erythropoietin - produced in the kidney
- stimulate production of red blood cells
BFU-E - burst forming unit erythroid
- insensitive to elecropoietin
- from large colonies after long incubation period
CFU-E - colony forming unit erythroid
- sensitive to erythropoietin
- form small clusters consisting of 8 to 32 cells
- target cells for erythropoietin

- found late in the erythroid maturation sequence

Eosinophils - derived from the bone marrow
- suppress the immunoinflammatory reaction and prevents
its spread
- phagocytize and kill invasive metazoan parasites
Basophils - exhibit chemotaxis and some phagocytosis
- contain blood histamine and degranulate to release
contents in
allergic reactions

Polyphyletic / Dualistic Theory

separate and distinct stem cell component for each of the blood cell

Regulation of Stem Cell Development Growth Factors and

Hormones

Pluripotential (CFU-S) Development

Humoral Agents Demonstrating Positive Effects on CFU-S

1. Phytohemagglutinin

2. Cyclophosphamide
3. Concavalin A
Postive Effects
1. Increase in CFU-S number
2. Increase in survival or protection from death
3. Increase in proliferation
Hydroxyurea - inhibitor of CFU-S proliferation

Granulopoiesis

production of monocytes and granulocytes (including neutrophils,

eosinophils and basophils)

Stimulating / Triggering Factors

1.
2.

Infection - main stimulus for the generation of large numbers of

neutrophils and monocytes
Colony Stimulating Factors - capable of promoting CFU-GM

proliferation
- responsible for the regulation of
granulopoiesis in vivo
- macrophage is an important source
Mechanism of Response to Microbial Invasion
1. Granulocytes and monocytes are recruited into and around the
infected tissue by release of chemotactic factors.
2. Granulocytes and monocytes stored in the marrow are mobilized
and
released into the blood.
3. Initiation of accelerated cellular production to satisfy the increased
demand.

Erythropoiesis

Mature RBC - maintain adequate supply of oxygen in tissues

- highly specialized end stage cell
Erythropoietin - single chain acidic glycoprotein
- augments erythropoiesis by stimulating the
production of new

erythroblastic cells to synthesize hemoglobin

Megakaryopoiesis

Platelets - originate from the megakaryocyte in the bone marrow

- play important role in hemostasis and development of
thrombosis
Thrombopoietin - increases platelet production
Megakaryocyte CSF - colony stimulating factor
- responds to number of bone marrow
megakaryocytes

Lymphopoiesis

Lymphocyte - provide for immune defense of the body

T Cells - also referred to as T lymphocytes
- mature in the thymus gland
- react directly with antigens to become specific cells
- responsible for reactions that produce:
1. delayed type hypersensitivity
2. graft rejection
3. tumor suppression
4. cellular immunity

B Cells - also referred to as B lymphocytes

- originate from the bone marrow
- interact with antigens to produce plasma cells
- also responsible for the production of antibodies and
immunoglobulins
- responsible for humoral immunity
Null Cells - non T, non B cells
Interleukin 2 - T cell growth factor
B Cell Activators
1. lipopolysaccharides
2. purified protein derivative
3. dextran sulfate
ERYTHROPOIESIS AND ERYTHROCYTE PHYSIOLOGY
Anton Van Leeuwenhoek - first described erythrocytes as small
round
Globules

Erythrocyte - primary function is to deliver adequate oxygen to

tissues
- has a life span of 120 days
BFU-E - earliest erythroid committed cell
- only few are normally present in bone marrow
- similar to medium sized lymphocytes
- reside in the null cell fraction of lymphocytes
- an increase of this has been demonstrated in some anemias
CFU-E - first to be identified
- closely related to rubriblast
- may resemble early erythroid but has larger nucleoli and more
mitochondria
Erythrocyte Growth Factor Stimulators
1.
2.
3.
4.
5.
6.
7.

Erythropoietin
Growth Hormones
Thyroid Hormones
Androgenic and Nonandrogenic Steroid Hormones
Insulin
Monocytes
Macrophages

Erythrocyte Growth Factor Inhibitors

1.
2.
3.
4.
5.

T Lymphocytes
Viruses
Interferon
Uremic Toxins produced by Renal Disease
Alcohol

Main Sites of Adult Hematopoiesis

1.
2.
3.
4.
5.

Vertebrae
Ribs
Sternum
Skull
Proximal Ends of Femur and Humerus

ERYTHROCYTE MATURATION
Rubriblast - first precursor recognized
- give rise to 16 mature erythrocytes through 4 cell
divisions which
takes 72 hours to complete
Nucleus - most important in the earliest stage of development
- site for DNA and RNA synthesis
- center for direction of cell development and maturation
Chromatin - composed of DNA, histones and other proteins
- finely dispersed and appears either condensed or granular
Heterochromatin - condensed of chromatin
- takes on dark blue color when stained with basic
dyes
Euchromatin - not well demonstrated
- does not stain well
Nucleoli - present in the rubriblast
- stain intensively with basic dyes
- contain RNA, proteins and a small amount of DNA
- involved in the production and distribution of RNA
Polyribosomes - clusters of ribosomes present in the cytoplasm of the
early
erythrocyte precursors
- site of synthesis of globin and other proteins

- attach to endoplasmic reticulum to form different

proteins
Golgi Apparatus - appears as a cluster od vesicles and plates of different
shapes
- referred to as traffic director for thousands of
protein
molecules being synthesized
- packing plant of the cell

Mitochondria - rod or oval shaped organelle

- serve as aerobic generator of energy for maturing cells
- helps in the insertion of ferrous iron into
protoporphyrin IX
during heme synthesis
Iron - present in the form of ferritin
- found freely dispersed in the cytoplasm or membrane bound in
vesicles
Ferritin - demonstrated by the use of Prussian Blue iron stain
Siderotic Granules - small blue aggregates present in Prussian Blue
staining
Ehrlich - first to describe nucleated erythrocyte precursors
Normoblasts - normal precursors
Megaloblasts - large abnormal precursors
- caused by the impairment of DNA synthesis
RUBRI
Rubriblast
Prorubricyte
Rubricyte
Metarubricyte
Reticulocyte
Erythrocyte

NORMOBLASTS
Pronormoblast
Basophilic Normoblast
Polychromatic
Normoblast
Orthochromatic /
Orthochomatophilic
Normoblast
Reticulocyte
Erythrocyte

ERYTHROBLAST
Proerythroblast
Basophilic Erythroblast
Polychromatic
Erythroblast
Orthochromatic
Erythroblast
Reticulocyte
Erythrocyte

Reticulocyte - also known as diffusely basophilic erythrocyte or

polychromatophilic erythrocyte
Stages of Development

Rubriblast

earliest erythrocyte precursor

size varies form 12 to 25 m
nuclear : cytoplasmic (N:C) ratio is high

nucleus usually occupies 80% of the cell

has a round to slightly oval shape
has dispersed fine clumps of chromatin and contains nucleoli
has a basophilic cytoplasm because of its high RNA content
easily mistaken as a myeloblast
gives rise to 2 prorubricytes

Prorubricyte

- measures 12 to 17m
- nucleus usually occupies 75% of the cell
- cytoplasm is basophilic
- golgi apparatus is usually visible in a light area near the nucleus
- chromatin is dark violet, coarser and more clumped than the
rubriblast
- nucleoli is absent
- divides 2 times, giving rise to 4 rubricytes

Rubricyte

measures 12 to 15 m
has a round nucleus and may be eccentric

nuclear chromatin is very coarse and condensed

may be confused as a lymphocyte
last cell division during maturation

Polychromasia - blue RNA mixed with red hemoglobin giving cytoplasm an

opaque violet-blue color
Early Rubricyte Cytoplasm - moderately polychromatophilic
Late Rubricyte Cytoplasm - have paler blue gray violet to slightly pinkish
color

last nucleated erythrocyte stage

measures 8 to 12 m
has a dense chromatin pattern
nucleus is pyknotic (dense mass of degenerated chromatin)
hemoglobin is the main constituent of the cytoplasm
nucleus is extruded at this stage

48

Metarubricyte

Reticulocyte

have irregular cytoplasmic borders

are retained in the bone marrow for 2 to 3 days before release to
marrow sinusoids
cell loses its organelles and assumes biconcave shape within 24 to
hours

Fibronectin - a glycoprotein in the bone marrow matrix

- as erythrocyte matures, it loses its ability to adhere to this
Reticulocyte Contents
1. Gologi Apparatus Remnants
2. Residual Mitochondria
3. Residual RNA

Mature Erythrocyte

approximately 7.2 m in diameter

it appears as a biconcave disc
presence of central pallor when stained in Wright stain
contains no mitochondria

Rubricyte & Prorubricyte

Plasma Cells
Have high N:C ratio
N:C ratio is low
Chromatin is fine to course but not
Has coarse chromatin
as coarse as in a mature plasma cell
Have eccentric nucleus and
perinuclear halo
Display intense and dark cytoplasm
Rubricyte & Metarubricyte
Chromatin pattern has more
clumped and lightened area
Has abundant cytoplasm
Cytoplasm is polychromatophilic to
pinkish in appearance

Lymphocytes
Has a dense homogenous
chromatin pattern
Usually have scanty cytoplasm
Cytoplasm has a distinct light blue
color
Nucleus tends to be more round

Structure and Physiology of the Mature Erythrocyte

Mature Erythrocyte - lacks nucleus and organelles
- hemoglobin is the main component

Shape and Deformability

Biconcave Disc Shape - the shape facilitates its oxygen and carbon
dioxide

transport function
- maximizes ratio of surface area to volume
- allows cell flexibility
Spheroid Shape - caused by membrane loss secondary to
fragmentation or
increased uptake of cations and water
Deformability - allow cell to adjust to small vessels in the
microvasculature
and still maintain a constant area:volume ratio

Membrane Composition

Three Basic Functions

1. Separate intracellular fluid of the cytoplasm to the extracellular fluid
of
the plasma.
2. Allow nutrient and ion passage selectively into and out of the cell.
3. Allow the cell to deform when required.
Phospholipids - arranged in a double layer called lipid bilayer leaflet
- the arrangement allows membrane to act as a liquid
sealer
Polar Head - oriented towards the aqueous environment
Nonpolar Fatty Acid Chain - face the hydrophobic leaflet interior

Unestrified Cholesterol - regulates membrane fluidity

- also regulates the membrane permeability to
electrolytes and non-electrolytes
98% of membrane cholesterol UNESTERIFIED
70% of plasma cholesterol ESTERIFIED
Proteins - bound to lipids throughout membrane
- vital to erythrocyte function
- characterized by the way they separate and form bands on
polyacrylamide gel
Peripheral Proteins - underline the lipid bilayer on the cytoplasmic side
- regulate shape and deformability
- allows membrane to have appropriate viscoelastic
properties
Important Skeletal Proteins
1. Spectrin (band 1 and 2)
2. Actin (band 5)

Integral Proteins - the glycoprotein designated for band 3

- an inorganic anion transport protein
- spans the lipid bilayer
- contains sialic acid which gives erythrocytes a
negative
charge
Enzymes - facilitate movement of substances and cofactors in and out of
the
cell
+
+
Na , K - ATPase - potassium pump
- controls active transport of sodium and potassium
Sodium = cell lysis
Potassium = cell shrinkage
Ca+2, Mg

+2

- ATPase - calcium pump

- moves calcium out of the cell to the plasma

against a
high concentration gradient
Calcium - involved in the regulation and stabilization of the membrane
phospholipid structure
Receptors - for molecules that are needed within the cell

Hemoglobin Viscosity

- normal erythrocyte hemoglobin concentration has low viscosity

- can cause cell to lysis when precipitated, polymerized, or
crystallized
- must be maintained in its reduced state to function properly
Methemoglobin - results from oxidation from normal ferrous state to ferric
state
- has no oxygen carrying capacity
Two Sites of Hemoglobin Molecule Prone to Oxidation
1. Iron atom in heme ring.
2. Sulfhydryl groups on globin chains.
Causes of Accumulation of Methemoglobin
1. Hereditary unstable hemoglobins.
2. Methemoglobin reductase deficiency.
3. Exposure to oxidant drugs.
Heinz Bodies - precipitated hemoglobin

- forms rigid cellular inclusions

- associated with various hematological disorders
- caused by oxidation of sulfhydryl groups

Energy Metabolism

maintain various components of erythrocyte

preserve membrane shape, enzymatic reactions and movement of
calcium, sodium, and potassium
reduce oxidized proteins

Glucose - principal energy source

- diffuse quickly without expense of energy
Pentose and Disaccharides - not metabolized by cell
Galactose - not normally metabolized by cell but have enzymes for its
degradation if it accumulates
EMP - Emben Meyerhof Pathway
- anaerobic method for energy generation though which glucose is
catabolized to lactate
- 90 to 95% of glucose is metabolized by this pathway
Important Intermediates in EMP
1. ATP - adenosine triphosphate
2. NAD+ and NADH - nicotinamide adenine dinucleotide
HMS - Hexose Monophosphate Shunt
- also known as Pentose Phosphate Shunt (PPS)
- aerobic method of energy generation
- 5 to 10% of glucose is metabolized with this
2, 3 DPG - 2, 3 diphosphoglycerate
- assists in modulating oxygen transport to the cell
- causes hemoglobin to resist oxygenation when there is low
oxygen
tension
- generated by the Rapoport Luebering Shuttle