Haematology Flashcards
What causes bleeding?
Under normal conditions the confinement of circulating blood to the blood vessels and maintenance of blood in a fluid state are dependent on maintaining a state of equilibrium between these processes. Loss of this balance may result in bleeding, which can be caused by:
1) Reduction in platelet number or function (primary haemostasis –platelet plug).
2) Reduction in coagulation factor(s) (secondary haemostasis – fibrin clot).
3) Increased fibrinolysis.
What are platelets?
Platelets are discoid, non-nucleated, granule-containing cells that are derived from myeloid stem cells. Platelets are formed in the bone marrow by the fragmentation of megakaryocyte cytoplasm and have a circulating lifespan of around 10 days.
How do platelets stick to damaged endothelium?
The plasma membrane contains glycoproteins (GPs) that are important for the platelet’s interactions. Following injury to the vessel wall platelets stick to the damaged endothelium, either directly to collagen via the platelet GPIa receptor or indirectly via von Willebrand factor (VWF), which binds to the platelet GPIb receptor.
What is the von Willebrand Factor (VWF)?
A glycoprotein that is synthesised by endothelial cells and megakaryocytes and circulates in plasma as multimers of different sizes. VWF mediates the adhesion of platelets to sites of injury and promotes platelet-platelet aggregation. In addition to its adhesive properties VWF is a specific carrier for factor VIII (FVIII).
What happens to platelets upon adhesion?
The adhesion of platelets causes them to become activated and changes their shape from a disc to a more rounded form with spicules to encourage platelet-platelet interaction.
What is ‘inside-out’/‘flip-flopping’?
A conformational change in the GPIIb/IIIa receptor, caused by platelet activation, to provide binding sites for fibrinogen.
What is the platelet release reaction?
The adhesion of platelets also initiates the release of the contents of their storage granules. There are two main types of ultrastructurally-identifiable granules: α-granules and dense granules. The platelet membrane is invaginated to form a surface-connected cannalicular system through which the contents of platelet granules are released.
What are the important components, of platelet granules contents, that are released?
ADP, fibrinogen and von Willebrand factor.
What is the role of fibrinogen?
Fibrinogen has a key role in linking platelets together to form the platelet plug.
What are platelets stimulated to produce, and why?
The prostaglandin - thromboxane A2 - from arachidonic acid that is derived from the cell membrane. Thromboxane A2, plays a role in platelet aggregation and is also a known vasoconstrictor and is especially important during tissue injury and inflammation.
Explain the action of antiplatelet drugs
Antiplatelet drugs are widely used for the prevention and treatment of cardiovascular and cerebrovascular disease. Aspirin and clopidogrel are the most commonly uses antiplatelet drugs:
1) Aspirin inhibits the production of thromboxane A2 by irreversibly blocking the action of cyclo-oxygenase (COX), resulting in a reduction in platelet aggregation. Although prostacyclin production is also inhibited by cyclo-oxygenase, endothelial cells can synthesise more COX whereas the non-nuclear platelet cannot. The effect of a single dose of aspirin therefore persists for around 7 days, until most of the platelets present at the time of aspirin ingestion have been replaced by new platelets.
2) Clopidogrel works by irreversibly blocking the ADP receptor (P2Y12) on the platelet cell membrane. Therefore the effect of clopidogrel ingestion also lasts for 7 days until new platelets have been produced.
What is ‘outside-in’ signalling?
Caused by fibrinogen binding to GPIIb/IIIa, which further activates the platelets.
What prevents inappropriate platelet aggregation?
The active flow of blood and the release of prostacyclin (PGI2) from endothelial cells; prostacyclin is a powerful vasodilator and suppresses platelet activation, thus preventing inappropriate platelet aggregation.
What role does positive feedback play in platelet recruitment, activation and aggregation?
The granular release of ADP and generation of thromboxane A2 have positive feedback effects resulting in further platelet recruitment activation and aggregation. They do this by binding respectively to the P2Y12 and thromboxane A2 receptor.
Describe the action of anticoagulant drugs
Anticoagulant drugs are widely used in the prevention and treatment of thrombosis. The main ones are:
1)Heparin - a mixture of glycosaminylglycan chains extracted from porcine mucosa. It works indirectly by potentiating the action of antithrombin leading to the inactivation of factors Xa and IIa (thrombin). Inactivation of thrombin requires longer chains of heparin chains, which are able to wrap around both the antithrombin and thrombin. It is administered intravenously or by subcutaneous injection. It is often used during cardiopulmonary bypass.
2) Warfarin - derived from coumarin, is a vitamin K antagonist that works by interfering with protein carboxylation. It therefore reduces synthesis of functional factors II, VII, IX and X by the liver. It is given as an oral tablet and its anticoagulant effect needs to be monitored by regular blood testing. Because it reduces synthesis of coagulation factors rather than inhibiting existing factor molecules, it takes several days to take effect.
3) Direct oral anticoagulants (DOACs) - orally available drugs that directly inhibit either thrombin or factor Xa (i.e. without the involvement of antithrombin). These do not usually require monitoring
What is prothrombin time (PT)?
It measures the integrity of the ‘extrinsic’ pathway. Blood is collected into a bottle containing sodium citrate (usually blue-topped), which chelates calcium, thus preventing the blood from clotting in the bottle
The sample is spun to produce platelet-poor plasma. A source of TF and phospholipid (usually a recombinant thromboplastin) is added to the citrated plasma sample, together with calcium to start the reaction; the length of time taken for the mixture to clot is recorded. The PT may be prolonged if there is a reduction in the activity of factors VII, X, V, II (prothrombin) or fibrinogen i.e. (‘prothrombin’ is a misnomer). When the PT is used to monitor vitamin K antagonist anticoagulant therapy such as warfarin, the results are expressed as the international normalised ratio (INR). This involves a correction for the different thromboplastin reagents used by different laboratories and means that all laboratories would be expected to obtain the same INR result for a given sample irrespective of the source of thromboplastin.
What is the activated partial thromboplastin time (APTT)?
It measures the integrity of the ‘intrinsic’ pathway. Performed by the contact activation of factor XII by a surface such as glass, or using a contact activator such as silica or kaolin. Contact activator, together with phospholipid, is added to the citrated plasma sample followed by calcium; the time taken for this mixture to clot is measured. Prolongation of the APTT is seen in a variety of situations where there is a reduction in a single or multiple clotting factors; in the the latter there may also be an associated prolonged PT. An isolated prolonged APTT (i.e. normal PT) is seen in patients with haemophilia A (factor VIII deficiency), haemophilia B (factor IX deficiency) and factor XI deficiency. However this may also be caused by factor XII deficiency which does not result in bleeding. (Note that FXII does not appear in the cell-based model described in ‘Coagulation (secondary haemostasis): formation of the stable fibrin clot’ and is not important for clotting in vivo).
What was the intrinsic-extrinsic cascade model?
An outdated model that has since been replaced by the cellular-based model. ‘Intrinsic’ refers to a system in which all components are in the plasma (factors XII, XI, IX, X and co-factors VIII and V), while the ‘extrinsic’ system comprises TF and factors VII, X, and co-factor V. It was believed that the extrinsic and intrinsic pathways ran in parallel, with initiation of the intrinsic pathway resulting from contact activation of factor XII. Through a greater understanding of factor XI and the recognition that people with inherited deficiencies of factor XII do not have bleeding problems, it became clear that the intrinsic-extrinsic model did not represent the physiological pathway of coagulation. However, the intrinsic-extrinsic model remains helpful in understanding blood tests used to assess coagulation.
What is the fibrinolytic system?
A mechanism to break down (lyse) clots. The principal fibrinolytic enzyme is plasmin, which circulates in its inactive zymogen form plasminogen. The activation of plasmin is mediated by tissue plasminogen activator (t-PA). However, t-PA does not activate plasminogen until these are both brought together by binding to lysine residues on fibrin. The breakdown of fibrin leads to the generation of fibrin-degradation produces (FDPs). Plasmin is not specific for fibrin and can also break down other protein components of plasma, including fibrinogen and the clotting factors Va and VIIIa. Plasmin is inhibited by antiplasmin which circulates in the blood.
Where are clotting factors synthesised?
Most clotting factors are synthesised in the liver. The exceptions to this are factor VIII and VWF, which are made by endothelial cells. VWF is also made in megakaryocytes and incorporated into platelet granules. Factors II (prothrombin), VII, IX and X are dependent on Vitamin K for carboxylation of their glutamic acid residues, which is essential for the function of these clotting factors. Factors V and VIII are co-factors.
What characterises each step of blood coagulation?
Each step is characterised by the conversion of an inactive zymogen (proenzyme) into an active clotting factor by the splitting of one or more peptide bones and exposure of the active enzyme site.
Where do clotting factors work and why?
Many clotting factors are believed to work on the exposed phospholipid surface of platelets, which helps to localise and accelerate these reactions.
What role do calcium ions play?
Calcium ions play an important role in the binding of activated clotting factors to the phospholipid surfaces of platelets.
What initiates coagulation?
The trigger to initiate coagulation at the site of injury is the tissue factor (TF) exposed on the surface of endothelial cells and leukocytes and on most extravascular cells in an area of tissue damage. TF is mainly located at sites that are not usually exposed to the blood under normal physiological conditions. As a result, blood only encounters TF at sites of vascular injury.
Outline the Initiation phase
TF binds to factor VIIa which leads to the activation of factors IX to IXa and X to Xa. This leads to the activation of prothrombin (factor II) to generate a small initial amount of thrombin (factor IIa). After thrombin generation has taken place, it is limited by the actions of protein C and its cofactor protein S to inactive factors Va and VIIIa.
Outline the Amplification phase
The small amount of thrombin, produced in the Initiation phase, mediates the activation of the co-factors V and VIII, the zymogen factor XI and platelets. Factor XI converts more factor IX to IXa, which in concert with factor VIIIa, amplifies the conversion of factor X to Xa.
Outline the Propagation phase
The amplification of the conversion of factor X to Xa, leads to a rapid burst in thrombin generation, which cleaves the circulating soluble fibrinogen to form the insoluble fibrin clot.
What is plasma?
Simply, it is defined as the liquid component of the blood, comprising around 55% of a given blood volume.
Outline the 6 functions of plasma
1) Clotting - clotting factors and von Willebrand factor, found in plasma, play keys role in blood clotting.
2) Immune defence - antibodies and complement proteins are found in plasma.
3) Osmotic pressure maintenance - proteins such as albumin help to maintain colloidal osmotic pressure.
4) Metabolism - nutrients such as glucose, amino acids and vitamins are transported in the plasma.
5) Endocrine - many hormones are soluble in plasma and following release into the blood, travel to their target organs.
6) Excretion - cell metabolism waste products such as urea are transported via the plasma fraction of the blood to the kidneys for removal.
Plasma makes up what percentage of body fluids?
Plasma makes up the second largest component of the extracellular fluids, making up around 7% of total body fluids and is the liquid component of blood.
What are the similarities between plasma and serum?
Both provide an easy to obtain clinical specimen that can be subjected to laboratory tests for diagnostic purposes. Elevated levels of some key molecules provide handy biomarkers of disease. A study found over 1,000 unique proteins in serum which were either secreted by cells or released into plasma during cell death.
What are the differences between plasma and serum?
Serum is generated by letting blood clot for several minutes, depleting the plasma of coagulation factors and trapping cells and platelets within the clot. Plasma is relatively quick to prepare whilst serum can generate a cleaner sample (containing few cells) but takes longer to generate. Plasma is produced when blood is collected in tubes that are treated with an anticoagulant, whereas serum supernatant is produced by centrifugation.
What is the main difference between plasma and interstitial fluids?
In composition, plasma is very similar to interstitial fluid with the main difference being that plasma contains much more protein.
Which is the most abundant plasma protein?
Serum Albumin
Outline the nature and function of Serum Albumin
It makes up around 55% of the total plasma proteins and is produced by the liver. It has several key functions including the transport of lipids, hormones and ions and maintaining the osmotic pressure of plasma. Fatty acids are released by lipolysis from the breakdown of triglycerides in adipose tissue and serum albumin plays a key role in transporting these around the body for use by other tissues in 𝛃-oxidation.
Globulins make up what percentage of plasma proteins?
Around 35% of plasma proteins. There are 3 main types: alpha globulins, beta globulins and gamma globulins.
Outline the function of alpha-1 globulins
Serum Alpha 1 globulins are represented by alpha-1 antitrypsin (A1AT) which is produced by the liver and enters the circulation where it plays an important role in inhibiting of enzymes which breakdown proteins (proteases). By inhibiting proteases, A1AT helps to protect tissues from a variety of enzymes, notably the protease neutrophil, elastase, released by neutrophils during inflammation. Defective or deficient A1AT can compromise the lung where degradation of lung tissue leads to a loss of elasticity and respiratory problems.
Outline the function of alpha-2 globulins
Alpha-2 globulins are typified by haptoglobin and 𝛂2 -macroglobulin. Haptoglobin binds to haemoglobin released from erythrocytes and the resulting haptoglobin-haemoglobin complex is removed by the spleen. Measuring levels of haptoglobin can be informative in the diagnosis of haemolytic anaemia in combination with other parameters. 𝛂2 -macroglobulin is a broadly active protease inhibitor which can inactivate fibrinolysis, the breakdown of fibrin involved in blood clotting.
Outline the function of beta globular proteins
These include the complement proteins C3 and C4. The protein transferrin is also a beta globulin, and as the name suggests plays a role in the transportation of iron. Generated by the liver, transferrin transports both dietary iron and that released from the stores of ferritin.
Outline the function of gamma globulins
Chief amongst the gamma globulins are immunoglobulins (antibodies) and the acute-phase protein C-reactive protein. Increases in the relative amounts of the gamma fraction of globulins can reflect increased immune system activity, associated with infection.
Electrolytes make up what percentage of plasma?
They make up only 1%, but are major contributors of osmolarity.
Outline the relationship between sodium and potassium both within the plasma and blood cells
Sodium is the most plentiful cation in the plasma and in contrast is found at levels 30 times lower inside blood cells. Conversely, the potassium levels are 30 times lower outside the red cell, in fact the relative concentrations of intracellular and extracellular sodium and potassium ions are almost inverted. To avoid being torn apart by differences in charge, the positive charge from the intracellular potassium is mainly balanced by extracellular chloride ions. The internally high concentration of potassium inside the cell is also neutralised by a variety of anions e.g. proteins, nucleic acids, phosphorylated proteins.
What is the function of sodium/potassium pump or sodium/potassium-ATPase?
The sodium/potassium pump maintains finely balanced electrolytes gradients, by the active transport of ions. This vital protein is also known as the sodium/potassium-ATPase, since hydrolysis of ATP is required to provide the energy for transportation. For every three Na+ ions that are actively transported out of the cell, two K+ ions enter the cell.
Why is the transport of Na+ and K+, so essential?
The significance of the active transport of Na+ and K+ is borne out by the fact that more than one third of the ATP consumed during rest is used to power the Na+-K+-ATPase. Maintaining the gradient of Na+ and K+ is key to the functioning of electrically excitable cells e.g. muscle fibres and neurones and also the maintain of cell volume.
What happens to cells when ATP levels are depleted?
Cells have a tendency to become more spherical because of the inward movement of sodium ions and water.
How can passive immunity be conferred from donor to patient?
As plasma contains immunoglobulins, the plasma of a patient recovering from a particular infection, is likely to contain relatively high levels of polyclonal antibodies which recognise the pathogen. If these are harvested and transferred to patients with the same infection, then passive immunity may be conferred from the donor to the recipient.
What is convalescent plasma?
Before the advent of vaccines, “convalescent plasma” was often used in clinical practice e.g. during the influenza pandemic of 1918. Nowadays, this is still used as a prophylactic treatment, taken post exposure to a variety of viruses, including hepatitis B and rabies.
Outline apherisis
From the Greek “to take away”, it is a technique in which the blood of a donor is collected and passed through a centrifuge to separate a particular cellular component, with the remained returned to the donor. In this way, different cell fractions can be purified for further use. The red cells comprise around 45% of the whole blood and are separated from the plasma by the buffy coat, which consists of leukocytes and platelets and makes up less than 1% of the whole blood.
Which disorders can therapeutic apherisis treat?
1) Plasma exchange - treatment if multiple sclerosis and myeloma.
2) Low Density Lipid Removal - treatment of patients prone to atherosclerosis
3) Red cell exchange - treatment of sickle cell disease
4) Platelet depletion - treatment of disorders of homeostasis
5) White blood cell depletion - treatment of leukaemia
Outline the concentrations of calcium ions inside and outside of cells
Calcium ions are found at intracellular levels several thousand times lower than those outside the cell. Increases in intracellular calcium are associated with signalling events and can be due to either the opening of calcium channels allowing influx form the exterior, or the release of intracellular stores. Intracellular magnesium is an important cofactor for many enzymes.
Proteins make up what percentage of plasma?
Proteins make up around 7% of plasma and can be assayed by the technique of electrophoresis.
What is haemopoiesis?
The formation and development of blood cells.
What cells count as red blood cells (erythrocytes)?
Granulocytes, monocytes and platelets, circulate in the blood and are produced in the bone marrow. They are ultimately derived from pluripotent or multi-potent hematopoietic stem cells (HSCs).
How are erythrocytes derived?
HSCs give rise to lymphoid cells and myeloid stem cells, from which red blood cells (erythrocytes), such as granulocytes, monocytes and platelets are derived.
How is haemopoiesis regulated?
A number of genes, transcription factors, growth factors (erythropoietin) and the microenvironment. Disruption of this regulation can disturb the balance between proliferation and differentiation, and may lead to leukaemia or bone marrow failure.
Outline the major function of erythrocytes
Their major function is oxygen transport and their approximate intramuscular lifespan is 120 days.
Outline the major function of neutrophil
It’s major function is defence against infection by phagocytosis and the killing of microorganisms. It’s approximate intravascular half lifespan is 7-10 hours.
Outline the major function of monocytes
It’s major function is defence against infection by phagocytosis and killing microorganisms. It’s approximate intravascular lifespan is several days.
Outline the major function of esinophil
It’s major function is defence against parasitic infection. Its intravascular lifespan is a little shorter than neutrophil (a few hours).
Outline the major function of lymphocytes
Their main function is humoral and cell immunity. Their approximate intravascular lifespan varies greatly.
Outline the major function of platelets
Their main function is Haemostasis. Their approximate intravascular lifespan is 10 days.
How many blood cells does the average person produce each day?
500 Billion
Outline the 2 essential characteristics of HSCs
1) Self renew: some daughter cells remain as HSCs, meaning that the pool of HSCs is not depleted.
2) Differentiate to mature progeny: other daughter cells (myeloid and lymphoid progenitor cells) follow a differentiation pathway.
Where are HSCs derived from in the embryo?
In Embryonic development, HSCs are derived from a layer called mesoderm. Primitive red blood cells, along with platelet precursors and macrophages are initially formed in the vadsculatureof the extra embryonic yolk sac, before the liver takes over between 6-8 weeks of gestation, as the main site of haemopoiesis. The liver continues to be the principal source of blood in the foetus, until shortly before birth, although the bone marrow starts developing hemopoietic actuitu from as early as 10 weeks gestation.
What becomes the sites of haemopoiesis following birth?
Following birth, the bone marrow is the sole site of haemopoiesis in healthy individuals. In children haemopoiesis occurs in almost all bones but by adulthood, this is restricted to the bone marrow of the pelvis, the vertebrae and the sternum, as well as the proximal ends of the long bones of the thigh (femur) and arm (humerus). However, other sites retain their ability to produce b,old cells if needed.
What is extra medullary haemopoiesis?
An increased hemopoietic drive, causing hemopoietic tissue to expand into other marrow cavities, which could lead to the development ofhaemopoietic foci, in the adult liver and spleen. This is called extra medullary haemopoiesis aas it occurs outside of the bone marrow.
How are HSCs and progenitor cells distributed in the bone marrow?
In an ordered fashion amongst mesenchymal cells, endothelial cells and the vasculature with which the HSCs interact.
What are haemopoietic growth factors?
They are glycoprotein hormones which bind to cell surface receptors. They regulate proliferation and differentiation of HSCs, as well as the function of mature blood cells. With the exception of erythropoietin which is produced in the kidney, haemopoietic growth factors are produced by cells of the bone marrow.
How is erythropoietin produced?
Red cells are produced under the influence of erythropoietin (EPO), which is synthesised in the cortical interstitial cells of the kidney, reduced oxygen supply to the kidney is a stimulus to erythropoietin synthesis.
How are granulocytes and monocytes produced?
Bone marrow production of granulocytes (neutrophils, acidophils, basophils and monocytes), is under the influence of multiple cytokines, such as the interleukins as well as the stimulating factors G-CSF and G-MCSF.
How are megakaryoctopiesis and platelets produced?
Under the influence of thrombopoeitin.
Outline the differentiation of lymphoid progenitors
B lymphocytes are first produced from the bone marrow, these cells make antibodies forming part of the humeral immunity response. T cells/lymphocytes, from the thymus, make cytokines and forming part of the cellular immunity response.
Outline the differentiation of myeloid progenitors
It produces erythroid, megakaryoctyes, platelets, granulocytes, neutrophils, basophils, acidophils and monocytes.
What is required for erythropoiesis?
Iron, Vitamin B12 and Folate. Low iron/vitamin B12/folate levels can lead to anaemia, either microcytic (iron deficiency) with areas of central pallor and smaller than normal blood cells, or macrocyctic (B12/folate deficiency) with larger blood cells that can grow but are unable to divide.
Outline erythropoiesis
Erythropoietin interacts with the erythropoietin receptor on red cell progenitor membranes and stimulates the bone marrow to produce more red blood cells. Hypoxia and anaemia both increase erythropoietin synthesis.
Outline the 2 main function of iron
1) It is essential for the synthesis of oxygen transport proteins Haemoglobin and myoglobin.
2) It is a key factor for protein and enzymes involved in energy, metabolism, respiration, DNA synthesis, cell cycle and apoptosis.
Why is iron essential?
It is essential for health skin, mucous membranes, hair and nails.
Outline the absorption of iron
The normal Western diet contains 10-20mg of iron per day. Iron is absorbed in the duodenum. Haem iron (animal derived: red meat, poultry and fish) is in the ferrous Fe2+ form, which is the best absorbed form. Non-Haem iron (plant and animal meat derived) is present mainly in the ferric Fe3+ form in food, which requires action of reducing substances (e.g. ascorbic avid and vitamin C) for absorption. Sources of no-Haem iron, such as soya beans, often contain phytates that reduce absorption.
Outline iron homeostasis
There is no physiological mechanism for regulating iron excretion, as iron can form free radicals that can damage body tissues. Iron overload is avoided by the tighter regulation of iron absorption in the gut by the hormone hepcidin. This allows iron absorption to be increased and stored alone or when there is a need for increased erythropoiesis, while minimising the risk of excess absorption when stores are inadequate. Iron in the plasma is bound to the transfer protein Transferrin, which delivers iron to the bone marrow for erythropoiesis. Iron is stored in the liver as the protein ferretin. Most iron is recycled and although is not actively excreted out of the body, a small amount is lost through the shedding of skin.
How do duodenal enterocytes alter iron absorption?
Hepcidin synthesis is suppressed by erythropoietic activity, this ensures iron supply by increasing ferroportin in the duodenum enterocyte, which increases iron absorption. When storage iron is high, hepcidin synthesis is increased, which binds and degrades ferroportin. This prevents the effluent of iron from the enterocyte, so it is lost instead when the cell is shed until the gut lumen.
How does hepcidin production in inflammatory states cause anaemia?
Hepcidin production leads to a reduction in iron supply. The resultant anaemia is called anemia of chronic disease.
Which cytokines are increased as part of the inflammatory response?
Interferon (IFNy) results in the reduction of erythropoiesis. IL-1 TNFalpha and IL-6 mediate the effect hepcidin. The increased production of pro-inflammatory cytokines in general, directly reduce the production of erythropoietin.
Outline Vitamin B12 and folate deficiency
DNA synthesis needs 4 immediate precursors, one of which is dTTP. Vitamin B12 (cobalamin) and folate are needed for dTTP synthesis, which itself is necessary for the synthesis of thymidine. B12 and folate deficiency affect all rapidly dividing cells: Bone marrow cells (can grow but are unable to divide properly), epithelial surfaces of the mouth and gut and gonads.
Where are Vitamin B12 and Folic Acid found in the diet?
B12 is found exclusively in food of animal origin, apart from fortified cereals. Folate is derived from many food sources, both animal and plant.
Outline the absorption of Vitamin B12
In the stomach, B12 combines with Intrinsic Factor (IF) made in the gastric parietal cells. In the small intestine, B12 binds to receptors in the ileum.
