Blood Sciences

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Blood Sciences I:

Clinical Haematology
Learning Outcomes
• Overview of clinical haematology investigations and
Haematopoiesis
• Describe the stages of red cell production
(Erythropoiesis)
• Describe the regulation of erythropoiesis
• Basic interpretation of red cell parameters
• Terms used to describe red cell morphology
• Abnormalities of the erthyron: Anaemia
Clinical Haematology
• Study of the numbers and morphology of the cellular elements of
the blood
➢ red blood cells (erythrocytes)
➢ white blood cells (leucocytes)
➢ platelets (thrombocytes)
➢ Fibrinogen
➢ Plasma or serum

• Haematologic analysis-Complete Blood count (CBC) also can be


known as Full blood count (FBC)

• CBC relevant for diagnosis of diseases of haematologic system but


also in surveillance and prognosis of other organ and systemic
diseases
Haemopoiesis (Haematopoiesis)
• Haemopoiesis: all aspects of blood cell formation.
• All blood cells originate from Haemopoietic stem cells
which reside in the bone marrow and circulate in the
blood
• The Pluripotent stem cell differentiates (commits) into
two lineages in the bone marrow: The myeloid lineage
and the lymphoid lineage
• The myeloid lineage gives rise to all myeloid cells –
erythrocytes, granulocytes (Neutrophils, Eosinophil's,
Basophils), monocytes and platelets.
• The lymphoid lineage gives rise to the development of T
and B lymphocytes .
Stages in Haemopoeitic Cell Development
▪ Haemopoiesis
1. Stem cell : Capacity for self renewal and proliferation.
2. Differentiation and Lineage selection ( specialisation)
3. Maturation: Non dividing cells will develop in mature cells
4. Functional mature cells
5. Cell death - apoptosis
Proliferation and Differentiation
Stem cells and Progenitor cells
• All precursor cells are derived from a common cell called the
Haemopoietic Pluripotent stem cell.
• Stem cells are relatively few (0.01% to 0.05% of the total
marrow population)
• Stem cells possess two characteristic features
• Their ability to proliferate to produce more cells and the ability for
self-renewal
• The haemopoietic stem cell has the potential to undergo
proliferation and develop into separate lineages (differentiation) and
eventually produce highly specialised cells which through the
process of maturation will develop into mature blood cells ; e.g.
erythrocytes, platelets, white cells
Haematology Definitions
• Progenitor Cell arise from stem cells in the bone
marrow and are only recognisable by cell culture
techniques. The progenitor cells will give rise to
microscopically recognised precursors cells. The
progenitor cells are referred to as Colony Forming Units
( CFU)
• Common Lymphoid Progenitor: a progenitor cell
capable of giving rise to all lymphoid cells.
• Common Myeloid Progenitor (also known as the
multipotent myeloid stem cell): a progenitor cell
capable of giving rise to all myeloid cells.
Haematology Definitions
• Colony: a group of cells derived from a single cell,
when progenitor cells are cultured in vitro.
• Colony forming unit (CFU): A progenitor cell which can
give rise to a colony of cells on Invitro culture. There
are also known as Colony forming cells CFC.
• CFU-G: A progenitor cell that can give rise to a colony
of granulocyte lineage when cultured in vitro.
• CFU-GM: A progenitor cell that can give rise to a
mixed colony of cells of both granulocyte and
monocyte lineages when cultured in vitro.
Haematology Definitions
• CFU-Mega: A megakaryocyte progenitor cell that can give
rise to a colony of cells of megakaryocyte lineage when
cultured in vitro. Megakaryocytes are the cells from which
platelets are produced.
• Burst forming unit erythroid (BFU-E): an earlier erythroid
progenitor cell which gives rise to a number of erythroid
colony forming units erythroid (CFU-E), which will
eventually result in the production of the mature red blood
cell
Haemopoiesis Essentials
▪ Haematological malignancies
( e.g. Leukaemia, Lymphoma ) generally are a result
of genetic mutations that affect the haematological
stem cell or any of its committed progeny, such as
the progenitor cells
▪ Stem cells can be harvested from the blood or
marrow and may be used to treat haematological
malignancies.
Stem Cell Hierarchy
Haematology Definitions
▪ CSF: Colony stimulating Factor; Haemopoietic Stimulating
or Growth Factors. An example is erythropoietin which is
the growth factor for red cell production.
▪ Cytokine: A group of low molecular weight soluble
proteins that acts as messengers within the immune
system and between the immune system and other
systems regulating processes such as Haemopoiesis,
inflammatory responses, wound healing. They include
interleukins, chemokine's and interferon's.
▪ Interleukin: is a cytokine secreted by one type of
leucocyte that has an effect mainly on other leucocytes.

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Haemopoiesis Model
▪ Most models of haemopoiesis include a number of
compartments
1. Pluripotent stem cell with the capacity for self renewal.
2. Progenitor cells which give rise to different lineages.
3. Progenitor cells gives rise to one lineage.
▪ Note: The above cells are identified by culture techniques
1. Precursor cells
2. Mature end cell compartment
▪ Note the precursor cells and the mature blood cells can
be microscopically identified in bone marrow and blood
samples. The precursor cells will eventually stop dividing
and the maturation process will result in mature cells.
Haemopoiesis
▪ In adult life Haemopoiesis predominantly occurs in the
bone marrow, the soft tissue within the centre of bones
where blood cells develop.
▪ In certain Pathological conditions Haemopoiesis may also
develop in other tissue, including the liver, lymph nodes
and spleen.
▪ Whereas in the foetus as well as the bone marrow
Haemopoiesis occurs in the liver, spleen and lymph nodes
during foetal development.
▪ In the embryo Haemopoiesis occurs in the yolk sac.
Haemopoiesis
▪ In healthy adults in the region of 5-10 x1011 blood cells are
produced in the bone marrow each day.
▪ Haemopoiesis is highly balanced and regulated and relies
upon the following
▪ Growth factors
▪ Cytokines
▪ Environmental factors
▪ Amount of oxygen in the body

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Stem cells and Progenitor cells

▪ Blood cell production


▪ Mature Granulocytes ->lifespan in bloodstream = hours
▪ Mature Erythrocytes ->weeks – months

▪ Increased Blood Cell Production


▪ Occurs when an increased number of cells is required in
response to increased demands i.e. blood loss and infection

▪ Bone marrow
▪ Microscopically Recognisable blood cells in various stages of
development as well as early precursors cells of lineages

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Bone Marrow Trephine Biopsy
Regulation of Haemopoiesis
▪ Haemopoietic growth factors are glycoprotein's hormones
that regulate the proliferation and differentiation of
haemopoietic progenitor cells and the function of mature red
cells.
▪ They regulate the balance between Haemopoiesis and
apoptosis ( ageing cells dying).
▪ The growth factors also react to external stresses such as
infection or blood loss.
▪ The growth factors are produced in different organs in the
body. They bind to surface receptors on target cells ( colony
forming units ) and can trigger replication, differentiation and
functional activation of their target.
Haemopoietic Growth Factors
▪ Haemopoietic Growth Factors or Colony Stimulating Factors (CSF)
are molecules involved in the stimulation, proliferation and
development of progenitor cells.
▪ EPO
▪ G (Granulocyte) CSF
▪ M (Macrophage) CSF
▪ GM-CSF
▪ IL (Interleukin) 3
▪ Thrombopoietin
▪ Stem Cell Factor
▪ VE (vascular endothelial growth factor)
▪ In contrast to classical hormones, haemopoeitic growth factors
and other regulatory cytokines are produced by several cell types
Haemopoietic Growth Factors
Factor Sites of Target Cells produced
Production
EPO Kidney CFU-E Red cells
GM-CSF Fibroblast CFU-GM Granulocyte
Macrophages Macrophages
Bone Marrow Monocytes
Sromal cells
Endothelial Cells
G-CSF As for GM-CSF CFU-GM Granulocytes
(neutrophils)
Thrombopoietin Liver cells CFU-Meg Megakaryocytes
Platelets

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Erythron
• Erythron: all red cells plus all pre-cursor red cell producing
tissues in an animal, including red cells in blood in blood
vessels and sinuses of spleen, liver and bone marrow

• Function: O2/CO2 transport between the tissues and the lungs


with haemoglobin as the carrier protein
Erythropoiesis
• This process requires an intact and integrated system
providing factors necessary for cell division , cell
maturation of erythrocyte precursors in the bone
marrow and to ensure the release of the cells into
the peripheral blood that will develop promptly into
mature red cells.
• The cells produced will provide a vehicle for the
transport of haemoglobin, which combines with
oxygen in the lungs and is delivered to the tissues.
• The cells in this system will also have to transport
carbon dioxide from the tissues to the lungs for
excretion.
• Cells should be able to survive in the circulation for
the duration of a normal life span (120 days)
Red Blood Cells
Maturation Phenomena
• The proliferation of blood cells occurs simultaneous with
differentiation and maturation .
• See diagram on maturation and development of red cells.
• All red blood cells precursors with the exception of the
late erythroblast are dividing cells.
• One pro erythroblast will possibly give rise to 16 mature
red blood cells.
• The late erythroblast will mature into a reticulocyte and
in turn into a mature red blood cell.
Maturation Phenomena
▪ As the red cell precursors mature in the bone marrow, not
only will you get a decrease in the size of the red cell
precursors, but you will also see change in the way the
cytoplasm stains and appears under the microscope. This
involves;
▪ Early normoblast- basophilic- ( dark blue)
▪ Intermediate normoblast-polychromatic ( lighter blue)
▪ Late normoblast- orthochromic ( light blue)
▪ Nucleus decreases in size and is eventually is extruded from
the mature red cell
▪ Red cell - Eosinophilic ( salmon pink) as the cell becomes
haemoglobinised and stains with eosin the acidic part of
the Leishman.
Reticulocyte
• Reticulocyte: a young erythrocyte newly released
from the bone marrow, into the peripheral blood
• It normally takes up to 48 hours in the blood stream
before the reticulocyte develops into a mature red
cell.
• Reticulocyte is a juvenile red cells that contain
ribosomal remnants and residual RNA from the
erythroblast precursors.
Stimulus for Red Cell Production
▪ Anoxia or hypoxia where there is less than the normal
concentration of oxygen in the blood and thus less available to
the tissues will act as a stimulus and plays a role in relation to
the control of red cell production.
▪ Thus erythropoietin increases in anaemia.
▪ The kidney organ when exposed to anoxia or hypoxia states,
triggers the production and release of the red cell growth
factor, erythropoietin.
▪ The erythropoietin stimulates erythropoiesis by increasing the
number of progenitor cells committed to erythropoiesis.

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Erythropoietin EPO
▪ Listed are some characteristics of the erythropoietin molecule.
▪ A Sialic acid Glycoprotein with alpha 1 globulin.
▪ 90% is produced in the kidney the remaining 10% is primarily been
produced in the liver.
▪ It is present in small amounts in normal plasma.
▪ The molecular weigh of human EPO is 34 kDa
▪ It is a glycosylated polypeptide of 165 amino acids with a distinct
Electrophoretic pattern
▪ Heat stable
▪ Produced in response to low oxygen tension (PO2). Reduced PO2 leads to
the release of erythropoietin from the kidney.

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Stimulus for Red Cell Production
▪ EPO stimulates RBC production which leads to an
increase in the number of red blood cells and the level of
haemoglobin available to carry oxygen to the tissues.
▪ EPO has been used by trainers to enhance performance
of horses.
▪ Animals living in high altitudes have a higher
concentration of EPO as they require more RBC to pick
up oxygen for the tissues.
▪ Note; a reduction in oxygen in the tissues leads to an
increase in erythropoietin. The erythropoietin acts on the
progenitor cells and the red cell precursors in the bone
marrow to increase the production of the red cells.

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Control of Erythropoiesis
▪ Erythropoietin will influence the process of cell
division in the red cell progenitor cells and the
red cell precursors cells ( the pronormoblasts and
the normoblast).
▪ The erythropoietin will effect receptors that will
signal and influence the level of DNA and mRNA
synthesis within the cell.
▪ Erythropoietin also effects the rate of transfer of
iron to the normoblast and the rate of haem
synthesis.

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Erythropoiesis continued
▪ Normal Erythropoiesis is termed normoblast.
▪ A Myelogram involves the examination the bone marrow
▪ In relation to red cell precursors the normal bone marrow
has the following proportion of immature red cells;
▪ Pro normoblast : 1%
▪ Early normoblast: 1-4%
▪ Inter. normoblast: 20-30 %
▪ Late normoblast: 70%

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Erythropoiesis continued
▪ Myeloid/erythroid ratio: ( MER) its ratio of immature
white cells to immature red cells in the bone marrow.
▪ Normal ratio is 4:1 (white : red)
▪ You have more white cell precursor cells in the marrow
due to the short life span of white cells in the blood.
RBC life span 120 days as opposed to a few days for
most of the white cells with the exception of the
lymphocytes.
▪ In leukaemia and in severe infection the ratio can
increase significantly (20:1 ratio).
▪ In severe blood loss or destruction of red cells you may
get a reduction in the ratio as there may be an increase
in the red cell precursors to boost production of red
cells.

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Erythropoiesis
▪ In a Vitamin B12 and or Folate deficiency, instead of
normoblast, the bone marrow produces Megaloblast.
(Large abnormal red cell precursors ). The bone marrow
is referred to as Megaloblastic.
▪ Hyper refers to over activity or over production of cells
in the bone marrow; Hyperplasic marrow.
▪ Hypo refers to a decrease in activity or underproduction
of the marrow; Hypoplastic marrow
▪ Aplasia or aplastic bone marrows means no production
of cells in the bone marrow.

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Variations from the Normal Maturation
Process in Erythropoiesis
▪ During blood loss or blood destruction, to increase the
production of red cells you get a shortening of the
intermitotic time ( speed of cell division) during the
proliferation and the maturation process.
▪ In iron deficiency anaemia where you get extra cell
division resulting in smaller microcytic cells.
▪ If you omit one or more of the mitotic steps you will a
larger than normal red cell. This occurs when you get in
Megaloblastic anaemia (due to B12 or Folate deficiency)
where you get a Macrocytic cells.

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Measurement of Erythropoieitic Activity

▪ Complete Blood Count (CBC)


▪ Blood film for red blood cell morphology
▪ Bone Marrow microscopy
▪ EPO assay. EPO levels may be low in severe renal
disease.
▪ Ferrokinetics; Use an isotope such as Fe59. Inject the
Fe59.This should be taken up by transferrin and
transported to the bone marrow. If there is normal
Erythropoeisis,the isotope should reappear a later in the
Haemoglobin Molecule in the red cells.

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Measurement of Erythropoieitic Activity
▪ Reticulocyte Count: A high reticulocyte count in the
blood stream would indicate an increase in the rate
of production of red cells.
▪ Blood volume. Red cell mass + Plasma Mass.
▪ Red cell lifespan studies: Use an isotope such as Cr51.
Take a blood sample off a patient and tag it with Cr51.
Inject the cells back into the patient. Measure the
lifespan of the injected cells. Should be 120 days.
▪ Haematinics studies which includes measuring the
levels of B12, Folate, and Iron.

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Bone Marrow Examination
• A bone marrow aspiration and a bone marrow trephine
biopsy are different procedures that often are done
together. The two procedures together are also referred
to as a bone marrow examination.
• Bone marrow is the spongy tissue inside your bigger
bones that produces red blood cells, white blood cells,
and platelets.
• Using a needle, the aspiration draws out a sample of the
liquid portion of your bone marrow. The trephine biopsy
also done with a needle removes a small, more solid part
of the bone marrow.
• The tissue is often taken from the back of your hip bone.
Bone Marrow Biopsy Needles

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