BIO3CBH – Blood Groups,

Transfusions and Platelets

Lecturer
Helen Irving
Intended learning outcomes

By the end of this topic, you should be able to:

•Assign blood types on the basis of routine blood tests

•Explain and provide solutions for haemolytic diseases of the newborn.
•Interpret outcomes from stem cell transplants
•Explain how platelets function in haemostasis
•Describe events occurring in coagulation and fibrinolysis
•Interpret blood test results on disorders such as thrombocytopaenia, and
explain your reasoning
•Provide rationale for use of specific blood products in care of chronic
wounds
BIO3CBH – Blood Groups
Lecturer
Helen Irving
Common blood groups and their frequency
• >400 red blood cell groups
• ABO followed by Rh systems most important
• Blood testing of donor is very important
• Blood typing is important for all transfusion recipients
but people who require regular transfusions are more
at risk
• Other groups important in transplants (stem cell (bone
marrow) in particular)

From Chris Bradley

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ABO blood antigens
• A glycosyl transferase protein
encoded from a single gene with 3
alleles (A, B and O) forms the
sugar residues defining the ABO
system.
• A and B alleles catalyse addition of
a or B terminal sugars.
• All people have the H antigen
except for those with the Bombay
phenotype (rare).
Source: Figure 30.2, Hoffbrand & Moss (2016) Hoffbrand’s essential Haematology 7th ed

Note that these antigens are also found in most body cells
ABO Antibodies
• Anti-A antibodies found in people with type B (IgM) and type O (IgG).

• Anti-B antibodies found in people with type A (IgM) and type O (IgG)

• No anti-A or anti-B antibodies found in type AB

• Group O are universal donors

• Group AB are universal recipients

 ABO grouping in patients
96 well plate test
• If patient is group A then red cells
suspended in saline agglutinate in
presence of anti-A or anti-A plus
anti-B serum
• Agglutination seen as a button of
cells in the bottom of the well.
• In negative reactions, cells are
dispersed and remain in suspension
• Anti-D is detecting Rh+ve cells

Source: Figure 30.3, Hoffbrand & Moss (2016) Hoffbrand’s essential Haematology 7th ed
Rhesus (Rh) system
• Rh blood group is composed of two related structural genes RhD and RhCE encoding
the membrane proteins carrying D, Cc and Ee antigens
• Rh groups are found only on red blood cells
• Rh antibodies rarely occur naturally and are thus the result of immune reactions to
prior transfusions or pregnancy
• RhD gene may be present (RdD+) or absent (Rh D-) – clinically most important
• Alternative splicing of RhCE gene generates two proteins encoding C or c and E or e
antigens (clinically potentially important in patients receiving multiple transfusions)

Gene D CcEe

Alternative
splicing
mRNA D E or e C or c
 Gel card technology for blood typing
• Gel card technology uses mini columns containing antibodies to various blood groups.
• A positive reaction is when the blood cells form a band in the gel. Negative is when
they go through to the bottom.
• Can buy cards for any blood group. Can be automated, accurate and less error.

http://www.palms.com.au/education/transfus/grouping.shtml
BIO3CBH – Blood Products
Lecturer
Helen Irving
Blood components for analysis or transfusion

https://www.memorangapp.com/flashcards/122533/Histology+of+blood+components/
 Preparation of blood components
https://pixabay.com/photos/blood-bags-red-red-blood-cells-91170/

Fresh frozen plasma (FFP)

for clinical use
Fresh Plasma
fro fractionation
FFP for • Albumin
• Gammaglobulin
Whole Blood • Specific antiviral
Cryoproducts
+/- Buffy coat immunoglobulins
leucodepletion • Anti-D
• Coagulation factors
Platelets

Red cell
concentrate

https://www.memorangapp.com/flashcards/122533/Histology+of+blood+components/
Blood products
https://pixabay.com/photos/blood-bags-red-red-blood-cells-91170/

• Blood stored at 4o C for up to 35 days.

• Leucodepletion: WBC <5 X 106. Reduces transfusion reactions and
alloimmunisation
• Packed Red Cells: Most transfusions.
• Washed red cells: washed in saline and reduces allergic reactions
• Autologous donation and transfusion in increasing demand
• Granulocyte concentrates: for severe neutropenia
• Platelet concentrates: platelet disorders
• Human plasma: Volume expander
• Factor concentrates
• Pooled immunoglobulin: valuable source of antibodies against common viruses
BIO3CBH – Blood product
transfusions
Lecturer
Helen Irving
Clinical significance of blood products and blood types
• Blood transfusions can save lives
• Whole blood
• Blood components

• Individuals who lack particular blood groups are likely to have

developed “non-self” antibodies to these groups
• The presence of these antibodies can lead to haemolytic transfusion
reactions
• Cross-matching and other pre-fusion tests to ensure patient receives
compatible blood
Haemolytic transfusion reactions

• Can be immediate (IgM) or delayed (IgG) and both are potentially fatal
• Immediate
• Transfusion of incompatible blood (usually ABO)
• Symptoms in minutes (fever, tachycardia, hypotension, possibly followed by renal
failure and disseminated intervascular coagulation (DIC))
• Urgently need to stop transfusion
• Delayed
• Infection – transmission of bacteria/parasites in blood
• Delayed transfusion reaction
• Iron overload
• Non-haemolytic reactions
• Usually due to antileukocyte antibodies in patient https://pixabay.com/photos/blood-bags-red-red-blood-cells-91170/
Transfusion of platelets and plasma/plasma products

• Platelet transfusion used to treat thrombocytopoenia

• Ideally matched at ABO/Rh with patient
• Repeated receipt can sensitise patient to HLA (MHC) class I antigens on the
platelet surface
• Plasma and plasma product transfusions
• FFP is used for many coagulation disorders including DIC, bleeding /
haemorrhage
• Various factor concentrates used to treat appropriate haemophilia
• Albumin (major plasm protein) and used for severe hypoproteinemia
• Immunoglobulins – can be specific and used in prophylaxis against infections
or to prevent haemolytic disease of newborn (anti Rhesus D )
BIO3CBH – Haemolytic
Disease of the Newborn
Lecturer
Helen Irving
Rh Haemolytic Disease of the Newborn (HDN) Plasma cells

1st Pregnancy Anti-Rh IgM

• Occurs in Rh-ve women with Maternal
Rh+ve mates circulation Rh-specific
• Initial pregnancy stimulated B cells
the mother to produce anti- Placenta
Delivery – mother
Rh from the exposure to Rh+ exposed to Rh+ve
cells that occurs during birth cells from baby
when the baby's and
mother's circulations mix

• Since anti-D can cross the

IgG
placenta, foetal cells in Haemolysis of
subsequent pregnancies are foetal cells by
haemolyzed by the mother's memory
cells Plasma mother's
antibody cells antibody

• Rh immune globulin (RhIG) 2nd Pregnancy

used to prevent sensitization
 James Harrison – anti-D in his plasma:
over 1000 blood donations, saved over 2 million babies
from Rh Haemolytic Disease
Slide from Chris Bradley
ABO Haemolytic Disease of the Newborn (HDN)
• More common than Rh HDN but milder.

• Poor development of ABO antigens on foetal red blood cells

• Reduced number of A and B antigens on foetal red blood cells

• Most commonly in group A or B babies born to group O mothers as they make

Anti A and B IgG that can cross placenta

• Jaundice treated by UV.

BIO3CBH – Stem Cell Transplants
Lecturer
Helen Irving
Autologous stem cell transplants
• Used for patients with haematological malignancies (e.g. Hodgkin’s or non-
Hodgkin’s lymphoma, multiple myeloma). Also sometimes for gene therapy of
a genetic disease
Patient Patient

Leucopheresis of stem
Bone marrow cells from peripheral High dose chemotherapy
from lilac crest OR blood after chemotherapy ± whole body irradiation
and G-CSF injections

± attempts to remove additional tumour

cells (e.g. monoclonal antibodies)
Intensive support
therapy
Stem cells infused
intravenously after storage Adapted from Hoffbrand & Moss (2016)
Allogenic typing for stem cell transplants
• Requires blood typing and also HLA (human leukocyte antigen or major
histocompatibility complex (MHC)) matching between donor and recipient
Donor Patient
HLA matched sibling
or unrelated donor

Bone marrow Leucopheresis of stem High dose chemotherapy

from lilac crest OR cells from peripheral ± whole body irradiation
blood after G-CSF
injections

±T-cell depletion
Intensive support
therapy
Donor stem cells
infused intravenously Adapted from Hoffbrand & Moss (2016)
Haplotyping human leukocyte antigen (HLA)
• The HLA system or major histocompatibility
complex (MHC) contains multiple genes that
are involved in directing T-lymphocyte
responses
• The individual’s diploid genomic DNA is
inherited from both parents and a goal of HLA
typing is to identify the haplotypes that are
present to better match donor and recipient
(the greater the HLA match the better the
transplant outcomes)
• Molecular techniques (Polymerase chain
reaction – PCR) are commonly used to
amplify and sequence specific gene regions
• Experimental chromosomal separation
techniques are in development along with
whole genomic sequencing approaches.
Murphy et al. (2016) Scientific Reports 6: 30381
 BIO3CBH – Platelets
Lecturer
Helen Irving

This presentation is about platelets and by the end of this presentation you should be able
to compare and contrast platelet formation with that of erythrocytes, granulocytes and
lymphocytes.

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 Haemopoiesis focussing on platelets

Source: Figure 1.2, Hoffbrand & Moss (2016) Hoffbrand’s essential Haematology 7th ed

As previously noted platelets are derived from the pluripotent common myeloid progenitor
cell (CFUGEMM) like erythrocytes and granulocytes. The circle highlights the platelet lineage
and how platelets are derived from the megakaryocyte progenitor cell.

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 Platelet production

Source: Figure 24.2, Hoffbrand & Moss (2016) Hoffbrand’s essential Haematology 7th ed

Platelets are derived from megakaryocytes in the bone marrow. Megakaryocytes are
interesting cells as they are polyploidy (i.e. contain many nuclei as they undergo
endomitotic synchronous nuclear replication and can become quite large as their name
suggests. Platelets are forming in the cytoplasm where numerous organelles are
entrapped. The larger and more mature megakaryocytes can release more platelets.

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 Megakaryocytes and platelets
• Platelets are derived from megakaryocytes
in the bone marrow.
• Megakaryocytes are polyploidy • Platelets in a blood film

From Chris Bradley

platelets

From Chris Bradley

P=platelets budding off from the cytoplasm.

The image on the left shows a megakaryocyte with several nuclei and platelets budding off
from the cytoplasm. The image on the right shows platelets in the typical blood film - note
that they are much smaller than an erythrocyte.

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 Growth factors responsible for haemopoiesis
Committed
progenitor cells Erythropoietin GM-CSF IL-3
Erythrocytes
IL-3 GM-CSF Thrombopoietin
Megakaryocyte Platelets

IL-1, IL-3, GM-CSF IL-3 M-CSF Monocytes

Monocyte-
IL-6, GM-CSF
granulocyte
GM-CSF, GM-CSF G-CSF IL-3
progenitor Neutrophils
Pluripotent SCF
stem cell GM-CSF IL-3 IL-5 Eosinophils

IL-3 Basophils

B lymphocytes

Interleukins (IL) 1, 2, 4, 6, 7, 12
Thymus T lymphocytes
SCF = stem cell factor, GM-CSF = granulocyte-monocyte colony stimulating factor, G-CSF = granulocyte colony stimulating factor, M-CSF = monocyte colony stimulating factor

The critical growth factors regulating development of platelets are: GM-CSF = granulocyte-
monocyte colony stimulating factor and thrombopoietin.

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 Regulation of platelet production
• Platelet production is regulated by thrombopoietin (TPO) which binds to a receptor
on the surface of megakaryocytes.
• TPO is produced by the liver after ”aged” platelets (lacking sialic acid) bind to the
Ashwell-Morell (formerly called asialic acid) receptor in the liver

Sialic aging
Platelet Galactose Platelet Galactose
acid

Aged platelets
bind to Ashwell-
TPO
Morell receptor
TPO stimulates production
platelet production

The diagram in this slide outlines the how the liver regulates platelet production by
producing thrombopoietin (TPO) which binds to a receptor on the surface of
megakaryocytes thereby stimulating release of platelets. Newly released or young platelets
have glycoproteins on their surface where the sugar chain terminates in a sialic acid
residue. As the platelets age, this sialic residue is removed. These ”aged” platelets (lacking
sialic acid) bind to the Ashwell-Morell (formerly called asialic acid) receptor in the liver and
a signal cascade results eventuating in the release of thombopoietin that is then able to
circulate to the bone marrow and stimulate platelet release.

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 Inherited platelet disorders (IPDs)
The 51 known genes
underlying IPDs. The
diagram depicts the
process of
megakaryopoiesis (MK)
and platelet formation.
HSC = haemapoietic stem cell

There are 51 genes underlying inherited platelet disorders. The diagram depicts the process
of megakaryopoiesis and platelet formation. The lists below the diagram are where the
known inherited platelet disorder genes are categorized according to their effect on
megakaryocyte and platelet biology.

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 BIO3CBH – Platelet Function
Lecturer
Helen Irving

This presentation is about platelet function and by the end of this presentation you
should be able to
1. Describe how platelet characteristics contribute to their function
2. Outline the process of haemostasis and describe the role of platelets in this process
3. Justify the roles of multiple adhesion factors

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 Haemostasis involves blood vessels, platelets and blood coagulation

Vessel injury

Collagen exposure Tissue factor

Platelet adhesion

Platelet activation Blood

Serotonin Shape change, granule secretion, etc. coagulation cascade

Vasoconstriction Thromboxane A2, ADP

Thrombin
Platelet aggregation

Reduced blood flow Primary haemostasis plug Fibrin

Secondary (stable)
haemostasis plug
Adapted from Hoffbrand & Moss (2016)

This diagram summarises events in haemostasis following vessel injury and highlighting
the critical role of platelets in plug formation. When vessels are injured, collagen one of
the most common proteins in the extracellular matrix is exposed and platelets in blood
adhere to the collagen and become activated indicated by their shape changes and
secretion of various factors such as serotonin , thromboxane A2, etc. This leads to
vasoconstriction and platelet aggregation as the primary haemostasis plug is formed.
The vessel injury also releases tissue factor and blood coagulation cascade is activated
releasing thrombin and fibrin that stabilise the primary plug and as more fibrin attaches
form the secondary stable plug to stop bleeding and allow the wound to heal.

Main function of platelets is the formation of mechanical plugs during the haemostatic
response to vascular injury
Adhesion involves immobilization at sites of vascular injury which is partly mediated by
von Willebrand factor (vWF)
Aggregation involves platelet:platelet interactions and this is again partly mediated by
von Willebrand factor (vWF)

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Blood vessel anatomy, endothelial cells and injury
• Endothelial cells are active in maintaining vascular integrity and form the basement
membrane separating proteins in the interstitial vascular connective tissue (e.g.
collagen, fibronectin, elastin) from circulating blood.

• Following injury, endothelial cells synthesise Tissue factor (TF). Endothelial cells also
synthesise von Willebrand Factor (vWF), factor VIII, plasminogen activator,
antithrombin, thromomodulin

Source: phweb.bumc.bu.edu/otlt/MPH-Modules/PH/PH709_Heart/PH709_Heart2.html

Lets start by looking at the blood vessels. The endothelial cells are active in maintaining
vascular integrity and form the basement membrane separating proteins in the
interstitial vascular connective tissue (e.g. collagen, fibronectin, elastin) from circulating
blood.

Following injury, endothelial cells are activated and have a very important role as they
synthesise tissue factor (TF). Endothelial cells also synthesise von Willebrand Factor
(vWF), factor VIII, plasminogen activator, antithrombin, thromomodulin. All of these
factors help regulate haemostasis .

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 Platelets are sticky – many adhesion molecules

• Platelets have adhesion molecules called

integrins on their surface enabling them to
adhere to each other and to vessel walls as
well as other adhesion ligands such as
• von Willebrand factor (vWF)
• Collagen
• Fibrinogen
• Laminin
• Thrombopoietin
• von Willebrand factor (vWF) and fibrinogen
From Chris Bradley are also important in platelet aggregation

The surface of platelets is coated in numerous glycoproteins. Many of these

glycoproteins are adhesion molecules called integrins. The surface adhesion molecules
on platelets enables them to adhere to each other and to vessel walls as well as other
adhesion ligands such as
von Willebrand factor (VWF)
Collagen
Fibrinogen
Laminin
Thrombopoietin
von Willebrand factor (VWF) and fibrinogen are also important in platelet aggregation

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 Platelets close up – transmission electron (TEM) micrographs

From Chris Bradley

Note: numerous organelles and membrane systems shown in platelet cross sections

If we look at platelets close up using transmission electron microscopy, we can see

numerous organelles and membrane systems are present inside the platelets. Platelets
actually contain multiple secretory vesicles and when activated release the contents of
these.

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 Platelets close up – scanning electron (SEM) micrographs

• Platelets at rest • Activated platelets showing

• Their resting shape is called lenticular pseudopodia.
• Ends of pseudopodia are dense with
adhesion receptors

From Chris Bradley From Chris Bradley

These scanning electron micrographs show the difference in shape between resting and
activated platelets. The resting shape of platelets is called lenticular as it is like a lentil.
The surface looks relatively smooth. Activated platelets look a bit like small dendritic
cells with pseudopodia – the ends of which are densely coated in adhesion molecules.
These allow the platelets to stick to each other and also the collagen at sites of blood
vessel injury.

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Platelet aggregation involves multiple adhesion receptor ligand interactions

The multiple adhesion receptor-ligand interactions that occur in platelet aggregation are
important as it helps allow the plug to form when blood is flowing fast in an artery for
instance. This is known as high shear flow. The initial tethering of platelets to the surface
of immobilized platelets involves vWF-GPIb integrin interaction. This adhesive
interaction is rapidly reversible and at high shear rates does not readily support stable
platelet-platelet adhesion, resulting in platelet movement (translocation) across the
thrombus surface. As Jackson describes, Platelet stimulation by one or more soluble
agonists during translocation promotes the binding of vWF and fibronectin to integrin,
leading to sustained platelet-platelet adhesion. At elevated shear rates, the principal role
of the fibrin(ogen)-integrin interaction is to stabilize formed aggregates and allow the
secondary plug to form.

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Platelet secretion reactions and amplification
• Following platelet activation, the contents of α-granules are released
including fibrinogen, factor V, von Willebrand factor, fibronectin, heparin
antagonist (platelet factor 4, PF4), PDGF (platelet derived growth factor), etc.
• PDGF stimulates multiplication of vascular smooth muscle cells
• Thromboxane A2 (TXA2) is formed new in activated platelets and acts to
alter signalling cascades to stimulate release reactions. Secreted TXA2 also
acts as a vasoconstriction agent
• Platelet aggregation and release stimulate the coagulation cascade.

• Note: Inhibitors of platelet activity

• Prostacyclin (PGI2) synthesised by vascular endothelium cells inhibits platelet
aggregation
• NO (nitric oxide) constitutively released from endothelial cells (also macrophages,
platelets) inhibits platelet activation and promotes vasodilation

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The end