Haematology and Transfusion Science (BMS 303)

Week 1 Haematopoiesis

Module Overview
Lectures, Thursdays 10.15 -13.15, LT16 Week 7 The White Blood Cell Week 8
Haematological malignancies

Week 2 The Red Blood Cell

Week 5 Haemostasis

Week 3 Haemoglobin

Week 6 Disorders of Haemostasis

Week 9 Myeloid Malignancies Week 10 Lymphoid Malignancies

Week 4 Nutrition & Blood

Week 11 Transfusion Science

Module Co-ordinator : Dr Declan McKenna

Module Practicals
Thursdays, 14.00- 17.00, TL1 Practical 1 Manual Blood Count Practical 4 White Blood Cell Differential Count Practical 5 Coagulation

Module Resources
All module resources located within BMS303 module area on BBLearn Module Handout Lecture Notes (New notes released each week) Coursework guidance Practical Handbook Self Assessment Quizzes (one per week) Reading List and electronic resources Past Papers Revision Materials (Final Week)

Practical 2 Osmotic Fragility Practical 3 Haemoglobin Estimation

Practical 6 Blood Grouping

Recommended Textbook(s)

Other Useful Resources

Journals Blood British Journal of Haematology Websites American Society of Haematology http://www.hematology.org/ Heme Team Interactive Haematology http://www.hemeteam.com/ Atlas of Haematology http://www.hematologyatlas.com/ Apps CellAtlas : Guide to Blood Cell Morphology

Essential Haematology Hoffbrand & Moss 6th Edition

Haematology Blann, Knight & Moore

Dacie & Lewis Practical Haematology Lewis, Bain & Bates 10th Edition

Whats so big about Blood?

Blood has immense significance in human culture Religious symbolism Bible, Quran, various religions sprinkling blood was called bledsian = blessing Inheritance: Blood relation, bloodline, blood is thicker than water, Royal blood, blue blood Conflict : blood feud, bad blood, blood bath, blood money Emotion : cold-blooded, hot-blooded, red-blooded, merriness (Sanguine) Symbolic : Blood brothers, Blood sacrifice, blood oath, signed in blood, Blood on hands Fiction Vampires I smell the blood of an Englishman! Gore/violence/movies/videogames/death/censorship

Blood in Medicine
Egyptians bathed in blood for health Greeks : One of the four humors (body fluids) Associated blood with springtime (sanguine) Believed heart contained the soul Believed circulatory system carried air Romans : drank blood In 2nd century AD, Galen developed a theory of the blood system that lasted until 1200s Arabia In 1242, Ibn al-Nafis realised blood passed from right to left side of body via lungs, not heart England In 1600s, William Harvey published his seminal account of the circulatory system, with heart as pump

Blood Components
In the 1600s, the invention of the microscope meant the discovery of blood cells Red Blood Cells (late 1600s) White Blood Cells (1800s) Platelets (1800s) Plasma could be separated from cells (early 1900s) Now, we know blood is a mix of cells (~45%) and plasma (~55%)

WHAT IS HAEMATOLOGY AND WHY IS IT IMPORTANT?

From the Greek haima, meaning blood
VIDEOLINK : A Day in the Life of a Haematologist http://www.youtube.com/watch?v=yIQz360XRZU

In the course of this module, we will focus on many of the areas mentioned in this video Red Blood Cells / White Blood Cells / Platelets Immune system Leukaemia / Lymphoma Coagulation Anaemia Blood Transfusion Blood Grouping

Week 1 HEMATOPOIESIS

Introduction
In Lecture 1 we introduce key concepts in haematology and transfusion science, many of which will be expanded on in later lectures. We discuss how haematopoietic stem cells are the foundation of the adult blood system, sustaining the lifelong production of all types of blood cell. We consider the problems caused by abnormal blood cell production and describe how stem cell transplantation can be a possible cure for haematological disease. We also introduce some basic laboratory techniques that are employed in the study of haematology

Content
Haematopoietic Stem Cells Haematopoiesis Abnormal Hematopoiesis Haematopoietic Stem Cell Transplantation Common Haematological Techniques Conclusion

Learning Outcomes
After completing this lecture you should be able to: Know the characteristics of Haematopoietic Stem Cells. Summarise the process of haematopoiesis (blood cell formation). Give examples of disorders where haematopoiesis is abnormal. Describe how haematopoietic stem cell transplantation works in the treatment of haematological disease. List the various haematological tests than can be employed in blood testing in the laboratory

Haematopoietic Stem Cells

Haematopoietic stem cells (HSCs) have two main characteristics:

1. Self renewal They are able to proliferate and form more stem cells, a process known as self-renewal. So although they are used to continually produce new blood cells, the overall stem cell numbers remain constant in a normal healthy individual. 2. Differentiation The second characteristic feature of HSCs is that they are pluripotent, meaning they are able to undergo differentiation to produce highly specialised mature cell types. There is also considerable amplification in the process with one stem cell capable of producing a million mature blood cells after 20 rounds of cell division

Self renewal and differentiation of HSCs As they mature, HSCs become increasingly differentiated and lose the ability to self-renew A single HSC can give rise to > 106 mature cells after several rounds of division

Sites of Haemopoiesis
Foetus 0-2 months 2-7 months 5-9 months yolk sac liver, spleen bone marrow

Sites of Haemopoiesis
Foetus
Yolk sac birth

Adult
Central skeleton

Haematopoiesis

(vertebrae, ribs, sternum) Liver & Spleen

Infants Adults

Bone Marrow (all bones) Bone Marrow (vertebrae, ribs, sternum, skull, sacrum, pelvis, ends of femurs)

Distal bones
1 3 5 7 9 10 20 30 40 50 60

Months

Years

Bone Marrow Environment Haematopoietic stem cells are located within the bone marrow The stem cell compartment. Ideal environment for stem cells to grow and develop. Bone marrow is composed of stromal cells and a microvascular network

Bone Marrow Structure

Stromal cells include: Macrophages Fibroblasts endothelial cells fat cells reticulum cells

They secrete extracellular molecules collagen fibronectin haemonectin laminin proteoglycans (growth factors and adhesion molecules)

HSCs are stored in the bone marrow which provides a suitable microenvironment for promoting both self-renewal and differentiation where appropriate.

Scanning electron microscope image of bone marrow

Bone marrow smear

Haematopoiesis
Haematopoiesis (also known as haemopoiesis) is the process whereby HSCs develop into mature blood cells via a series of committed progenitor cells that are restricted in their developmental potential. The progeny of pluripotent stem cells become more irreversibly committed to specific cell lineages with each cell division. The further along the differentiation pathway a cell progresses the more restricted the range of mature cells it can produce. As they mature, fully differentiated blood cells are gradually released from the bone marrow stromal environment into the bone marrow microcirculation and eventually into the blood circulation. Among the earliest steps in haematopoiesis is the commitment and differentiation of the haematopoietic stem cell to become the precursor for cells of either the myeloid lineage or the lymphoid

B o n e

Figure explanation (previous slide) Representation of the pluripotent HSC and the various progenitor and mature cells that arise from it. The various progenitor cells can be identified in vitro by culturing in semi-solid medium and observing which type of colony they form (CFU). Abbreviations: CFU, colony forming unit; BFU, burst forming unit; Baso, basophil; E, erythroid; Eo, eosinophil; GEMM, granulocyte, erythroid, monocyte and megakaryocyte; GM, granulocyte, monocyte; Meg, megakaryocyte; NK, natural killer.

BFU-E

M a r r o w Pluripotent stem cell

Myeloid stem cell

BFU-E

CFU-E
CFU-E

CFU-E
B l o o d

These two pictures were taken in a lab in the University of Ulster and show the early stages of erythroid differentiation using in this case embryonic stem cells.

Erythrocyte

BLASTS
In vivo, immature cells in the various pathways are known and identified as blasts, of which there are different types, depending on which lineage they belong to. For example : Myeloblast : Lymphoblast: Erythroblast: blast cell from myeloid pathway blast cell of the lymphocyte pathway blast cells in the red blood cell pathway.

Mature blood cells produced via the myeloid and lymphoid lineages
Myeloid Lineage (CFU GEMM progenitor cell) Red cells (Erythrocytes) Platelets Monocytes Neutrophils Eosinophils Basophils Lymphoid lineage (Common lymphoid progenitor cell (CFUL)) B-Lymphocytes T-Lymphocytes NK Cells

These terms and others become important when studying haematological disease, since an increase in blast numbers and/or change in location from bone marrow can be indicative of disease. This will be discussed further in Lectures 8 to 10.

Regulation of Haematopoiesis
The cell type that a stem cell matures into is largely decided by the external signals it receives. Haematopoietic Growth Factors (HGFs) play a critical role in regulating the proliferation and differentiation into various mature blood cells These can act synergistically or may induce the production of one another by their action upon cells in the bone marrow environment. The figure on next slide shows major growth factors involved in haematopoiesis of myeloid lineages. Abbreviations: SCF, stem cell factor; IL, interleukin; GM-CSF, granulocyte-macrophage colony stimulating factor; M-CSF, macrophage colony stimulating factor; EPO, erythropoietin; TPO, thrombopoietin; G-CSF, granulocyte colony stimulating factor.

MYELOID

LYMPHOID

HGF EFFECT ON MYELOID LINEAGES

Characteristics of Growth Factors

Glycoproteins that normally act at very low concentrations Usually produced by many cell types e.g. stromal cells, monocytes, macrophages and lymphocytes Usually affect more than one lineage Usually show synergistic or additive interactions with other factors Multiple actions Biological effects mediated via specific receptors.

Important Growth Factors

Many HGFs have been identified in the past decades and while the function and role of many are well characterised and understood, the actions of some growth factors and how/when/why they act is still unclear. Some of the better understood HGFs are listed below. Stem cell factor (SCF) Interleukin-3 (IL-3)

Figure 6. Growth factors may stimulate proliferation of early progenitors, direct differentiation, stimulate maturation, suppress apoptosis of control function of mature cells, as exemplified above for the action of G-CSF upon early progenitor cells and mature neutrophil

Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF)

Erythropoietin (EPO) Discussed further in Lecture 2 Thrombopoietin (TPO) Discussed further in Lecture 5 Macrophage-Colony Stimulating Factor (M-CSF) Granulocyte-Colony Stimulating Factor (G-CSF) The link below has vast amounts of additional information about HGFs and cytokines. http://www.copewithcytokines.de/cope.cgi

Haematopoietic Growth Factor Receptor Signalling The multiple actions of HGF on haematopoietic cells are mediated through recognition of the growth factor by its specific cell surface receptor. Most haematopoietic cells have only a few hundred receptors per cell for each growth factor and low levels of growth factor binding to their specific receptor causes important biological responses. This is achieved via a series of intracellular signalling mechanisms. Binding of a growth factor to its cell surface receptor leads to the activation of associated kinases and the phosphorylation of the receptor. There are many varied and complicated signalling mechanisms activated downstream of growth factor receptors. A few of the main signalling pathways are shown on the next slide.

Figure 7. Binding of a growth factor can activate intra-cellular signalling pathways which will ultimately lead to the transcriptional activation of specific genes, which promote cell growth.

Mature Blood Cells

Successful hematopoiesis results in the production of several types of mature blood cell, each of which has a particular function within the body (see Table on Next Page). A healthy individual will have cell counts within the reference ranges shown, although age, sex and general health must also be taken into account in interpreting results. Any deviation outside these values may be indicative of an underlying illness or abnormality, which may range from a simple infection to a more serious blood disorder. We will discuss Red Blood Cells (erythroblasts) and their function in Lectures 2 & 3 Platelets and their function in Lecture 5 & 6 White Blood Cells and their functions in Lecture 7

TYPE

LINEAGE

NAME Erythrocytes Platelets Monocytes

PRODUCTION PROCESS Eryrthopoiesis Thrombopoiesis

MAJOR FUNCTION

TYPICAL NUMBERS
5.0 0.5. x 1012 / L (Men) 4.3 0.5. x 1012 / L (Women)

Red Blood Cells Platelets

Carry oxygen (and CO2) round the body (Haemoglobin) Blood Coagulation
Become tissue macrophages which phagocytose and digest invading micro-organisms and foreign bodies Immune response (can release histamine and serotonin) Modulate allergic inflammatory response and destroy larger parasites Phagocytose and destroy invading bacteria Immune response (Make Antibodies) Kill virus-infected cells and regulate activities of other white blood cells Kill virus-infected cells and some tumour cells

280 130 x 109 / L

Monopoeisis

0.2 - 1.0 x 109 / L

Myeloid

Basophils

Granulopoiesis

0.02 0.1 x 109 / L

White Blood Cells

Eosinophils

Granulopoiesis

0.02 0.5 x 109 / L

Neutrophils B-Lymphocytes

Granulopoiesis Lymphopoiesis

2.0 7.0 x 109 / L

Lymphoid

T-Lymphocytes NK Cells

Lymphopoiesis

1.0 3.0 x 109 / L (total lymphocytes)

Lymphopoiesis

VIDEO LINK. How Blood cells are formed http://www.youtube.com/watch?v=tDTLC2swhlQ&feature=related VIDEOLINK. What is blood? http://www.youtube.com/watch?v=CRh_dAzXuoU&feature=related

Aplastic Anaemia
Abnormal Hematopoeisis can lead to a variety of disorders Aplastic anaemia arises from a reduction in the number of HSCs a failure of remaining HSCs to divide and differentiate sufficiently exposure to radiation, chemicals, drugs and viruses. Aplastic anaemia results in pancytopenia. Pancytopenia is the reduction in the blood count of all the major blood cell types; red, white and platelet. This is characteristic of aplastic anaemias, although it can also be observed in various leukaemias.

Because they lack all types of blood cell, aplastic anemia sufferers may exhibit Fatigue, pallor (due to lack of red blood cells) Increased bruising / haemorrhage (due to lack of platelets for coagulation) Increased risk of infection (due to lack of white blood cells) Diagnosis involves a complete set of blood counts and tests, in order to distinguish cause and severity. Treatment and management may involve a number of approaches, including stimulating immune system, steroids and hormone therapy. However, the only chance of permanent cure will be a stem cell transplant.

Read more about aplastic anaemia here. http://www.mayoclinic.com/health/aplastic-anemia/DS00322

Hypoplastic Bone marrow in aplastic anaemia sufferer

HAEMATOPOIETIC STEM CELL TRANSPLANTATION

In many haematological disorders, a possible chance for a complete cure comes from haematopoietic stem cell transplantation (HSCT). This refers to the transplantation of blood stem cells into a patient and is often referred to as a bone marrow transplant (BMT), since the HSCs used for transplantation will have been collected from the bone marrow. However, it is becoming more common for HSCs to be collected from the peripheral blood, in which case the process is referred to as peripheral blood stem cell transplantation (PBSCT).

Sources of HSCs
The source of the HSCs can be : Autologous (From patient) Allogeneic (From matched donor). Matching involves ABO blood typing and HLA (tissue antigen) typing to minimise risk of transplant rejection. A sibling is the preferred donor as the likelihood of HLA-matching is higher Syngeneic (From an identical twin) This would be the ideal transplant.

ALLOGENEIC HSCT
The process of HSCT is usually as follows : 1. HSCs collected, isolated and stored from donor. 2. Patient is treated with highdose chemotherapy and/or radiotherapy to eradicate the patient's malignant cell population and eliminate the patient's bone marrow stem cells 3. HSC transplant to replace bone marrow stem cells 4. Blood system is restored by the healthy, transplanted HSCs

AUTOLOGOUS HSCT
In autologous HSCT the process is the same, except that the source of the HSCs is the patient themselves. In this case, the HSCs are harvested before the chemotherapy.

Autologous v Allogeneic HSCT

Source of HSCs Benefits Limitations HSCs may have potential to turn cancerous again No GVL Possibility of rejection (GVHD)

Sources of HSCs for transplant

Sources Bone marrow Peripheral blood Umbilical cord blood

Patient (Autologous)

No rejection (GVHD)

Donor (Allogeneic)

GVL

Graft versus Host disease (GVHD) is a complication that can occur after a stem cell or bone marrow transplant in which the newly transplanted material attacks patients body, even though the donor has been matched to the patient to minimise this. It can be acute (within 3 months) or chronic (may last lifetime). Graft versus leukaemia (GVL) (AKA graft versus tumour) is a similar effect, but is beneficial, in which any white blood cells in transplant mount an immune response against any residual leukaemic cells still present in the patient.

Bone Marrow
stem cells are harvested directly from crest of the ilium (pelvis) Has large amount of red marrow may also be taken from the sternum usually under general anaesthesia. minimally invasive procedure with only minor discomfort However, if patient cancer was due to stem cell defect, then using their own bone marrow may not be useful as disease will likely develop again

Harvested cells must be purified before storage for use.

VIDEO LINK -BONE MARROW TRANSPLANT http://www.youtube.com/watch?v=GIy2nMnuGGI

Peripheral blood
now the most common source of stem cells for HSCT. PBSC yield boosted by injections of G-CSF mobilizes HSCs from the donor's bone marrow into the peripheral circulation. Cells collected through a process known as apheresis. Donor's blood is withdrawn through a needle in one arm & passed through machine that removes WBC The red blood cells are returned to the donor.

Umbilical cord blood

Stem cells harvested from a newborn's umbilical cord and placenta after birth. Cord blood has a higher concentration of HSCs than is normally found in adult blood. Allogeneic cord blood is stored frozen at a cord blood bank because it is only obtainable at the time of childbirth Cord Blood banking (private and public) is increasingly big business in the US and UK. http://www.nhsbt.nhs.uk/cordblood/ However, numbers of stem cells in cord blood are small need ways to expand them in the lab. VIDEO LINK: How to collect cord Blood http://news.bbc.co.uk/1/hi/health/8556741.stm

Response to HSCT
Ideally, the patient will start to respond to treatment within a few weeks, which will be monitored by blood cell counts. Various supportive therapies may be administered before and during this post-transplant period to improve the chances of a successful outcome. These often include : platelet transfusions to prevent bleeding/haemorrhage. cyclosporin A therapy, which suppresses the patients immune system and therefore minimises the risk of transplant rejection. Red blood cells to aid oxygen transport.

However, although advances have been made in the administration of HSCT, the 100 day transplant related mortality (TRM) is still between 10 and 15%, dependent on patient age, donor type, disease status and stem cell source. Even in a successful transplant, patients can often experience many side-effects and it can take 1 -2 years for the treatment process to be entirely completed and full health restored. You can read some personal accounts of the experiences of various non-Hodgkins Lymphoma patients who received HSCT as treatment at the following website http://www.nhlcyberfamily.org/stories.htm

Complications of HSCT Many complications of SCT exist, which can be life-threatening if severe enough Graft failure Acute GVHD Chronic GVHD Organ toxicity Infection Skin reactions Bleeding Infertility Nevertheless, at present it offers one of the best chances for a complete cure from leukaemia / lymphoma and is increasingly an option for treating these diseases
Causes of death after SCT (2001-2006)

Common haematology techniques in the laboratory

Common haematological techniques

Blood Collection Blood counts (Practical 1, Discussed in Lectures 2 & 7) Blood films (Practical 4, Discussed throughout module) Flow cytometry (Discussed throughout module) Coagulation Screens (Practical 5, Discussed in Lectures 5 & 6) Other Tests
A great number of tests and assays can be performed for various micronutrients, components and characteristics of blood, as well as a range of measurements that fall into the field of clinical biochemistry (e.g. hormones, metabolites, lipids). A comprehensive overview of reference ranges for various tests can be viewed here. http://pathcuric1.swmed.edu/pathdemo/nrrt.htm. Note that different labs often have slightly different ranges and these will be constantly reviewed and updated where appropriate.

Blood collection

Blood is collected by a trained phlebotomist using venupuncture (ie from a vein). The blood is collected in tubes which have different coloured tops, indicating the presence (or absence) of various anti-coagulants to prevent the blood clotting. Without anti-coagulants, the blood will clot within 2-5 minutes and can be centrifuged to separate the serum from the cells For blood counts, EDTA is used to chelate the calcium ions that are essential for clotting, thereby preserving the integrity of the blood cells. For coagulation tests, however, sodium citrate is used to anticoagulate the plasma component. The correct collection and storage of the collected blood is crucial, since many tests become less reliable if the blood is handled or stored wrongly

Blood Counts
A full blood count is the most commonly performed haematological blood test Gives information on the numbers of each type of blood cell in a given sample Also measures haemoglobin concentration and calculates various Red Cell indices will be calculated . White cells differential count will indicate if the white blood cells are in the correct proportion In modern laboratories, this is usually carried out by automated analyzers and if the values fall outside normal ranges, it will be flagged up for further investigation. However, counts can also be performed manually

Blood films
Although blood counts are now usually automated, blood slides will also be produced if necessary to allow examination and/or count of the blood cells under a microscope. For routine slide analysis, this involves staining with specific dyes to highlight the distinctive features of each cell type. Romanowsky stains are universally employed for routine staining of blood films and when prepared properly gives very satisfactory results. Most modern haematology labs have machines that produce slides automatically. However, they can also be prepared by hand http://www.youtube.com/watch?v=ZyU9iZ9d9QI&feature=related

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Cell Stains
These stains are able to show subtle differences in shades of staining and stain granules differently due to the presence of two components azure B (trimethylthionin) and eosin Y (tetra-bromo-fluoroscein). Azure B is a basic dye and binds anionic molecules. Therefore, it targets acidic groupings of nucleic acids, proteins and primitive cytoplasm Eosin Y is an acidic dye and binds to cationic sites on proteins. Therefore, it targets basic residues, such as those found on haemoglobin The combination of these two elements binding can therefore be used to identify most cellular elements. Normal and/or abnormal cells can be thus identified by a trained biomedical scientist or clinician
Different types of blood cell can be identified in a blood smear, based on physical characteristics such as size, shape, colour, nuclei and granulation following staining with Romanowsky solutions. E, erthyrocyte (many); P, platelet (small); N, neutrophil; B, basophil; Eo, Eosinophil; M, monocyte; L, lymphocyte.

Flow cytometry
Immunophenotyping (flow cytometry) is a method whereby the physical and/or chemical characteristics of up to thousands of cells in a sample can be analysed in seconds. It is routinely used in the diagnosis of health disorders, especially blood cancers. HSCs, the various progenitor cells and mature blood cells all exhibit different patterns of marker proteins on their surface, known as Cluster of Differentiation (CD) markers. The CD marker profile of these cells is now well established, allowing them to be distinguished from each other. We can use fluorescent antibodies targeted to specific CD markers to label cells of interest. This means that flow cytometry can then be used to calculate how many cells displaying that marker (or a combination of markers) are present, based on fluorescent signals from the antibodies.

Flow cytometry simple example

The graph on the left demonstrates how different types of blood cell can be differentiated between, based on size (x-axis) and internal complexity (yaxis

Other common tests

TEST Erythrocyte sedimentation rate Plasma viscosity FUNCTION

thick

Assess inflammatory response to injury/treatment Measures how thick or thin the plasma Measures concentration of micronutrients and plasma proteins. E.g. Iron, ferritin, vitamin B12 or folate. Discussed further in Lecture 4 Separation methods like electrophoresis or chromatography are used to identify diseases linked to abnormal haemoglobin production . Discussed further in Lecture 3. Analysis of genetic information has improved. These techniques will be discussed further throughout the other lectures.

thickcomponent of blood has become

Haematinic assays Haemoglobin variant detection

The two graphs above compare the lymphocyte profile in blood from a normal person and a leukaemia patient. The leukaemia patient has an abnormal profile, indicating presence of large (ie toward right of graph), but undifferentiated (ie toward bottom of graph) lymphocytes, suggesting a problem with the differentiation and development of the lymphoid lineage. This information, along with other tests, helps make an informed diagnosis of disease.

L3

Molecular techniques

PCR, microarry

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CONCLUSION
This lecture has introduced several key concepts in haematology and transfusion science. We have looked at the concept of haematopoietic stem cells and how they can give rise to progenitor cells which will ultimately differentiate into mature blood cells. We have seen how this process of haematopoiesis is regulated by various growth factors, which help determine the lineage along which a stem cell will differentiate. Weve appreciated how haematological diseases can arise if haematopoiesis is disrupted and seen how stem cell transplantation may provide a cure for such diseases. Finally, we have reviewed commonly used haematological techniques used in blood testing. Throughout the remainder of the module we will return to these concepts and expand upon them in relation to various aspects of haematology.

WEBSITES ABOUT HSCs NIH Stem Cell Information http://stemcells.nih.gov/info/scireport/chapter5.asp National Cancer Institute Stem Cell Transplantation http://www.cancer.gov/cancertopics/factsheet/Therapy/bonemarrow-transplant Stem Cell Database http://stemcell.mssm.edu/v2/introduction.shtml

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