Haematology Flashcards
(213 cards)
1
Q
What causes bleeding?
A
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.
2
Q
What are platelets?
A
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.
3
Q
How do platelets stick to damaged endothelium?
A
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.
4
Q
What is the von Willebrand Factor (VWF)?
A
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).
5
Q
What happens to platelets upon adhesion?
A
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.
6
Q
What is ‘inside-out’/‘flip-flopping’?
A
A conformational change in the GPIIb/IIIa receptor, caused by platelet activation, to provide binding sites for fibrinogen.
7
Q
What is the platelet release reaction?
A
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.
8
Q
What are the important components, of platelet granules contents, that are released?
A
ADP, fibrinogen and von Willebrand factor.
9
Q
What is the role of fibrinogen?
A
Fibrinogen has a key role in linking platelets together to form the platelet plug.
10
Q
What are platelets stimulated to produce, and why?
A
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.
11
Q
Explain the action of antiplatelet drugs
A
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.
12
Q
What is ‘outside-in’ signalling?
A
Caused by fibrinogen binding to GPIIb/IIIa, which further activates the platelets.
13
Q
What prevents inappropriate platelet aggregation?
A
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.
14
Q
What role does positive feedback play in platelet recruitment, activation and aggregation?
A
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.
15
Q
Describe the action of anticoagulant drugs
A
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
16
Q
What is prothrombin time (PT)?
A
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.
17
Q
What is the activated partial thromboplastin time (APTT)?
A
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).
18
Q
What was the intrinsic-extrinsic cascade model?
A
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.
19
Q
What is the fibrinolytic system?
A
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.
20
Q
Where are clotting factors synthesised?
A
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.
21
Q
What characterises each step of blood coagulation?
A
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.
22
Q
Where do clotting factors work and why?
A
Many clotting factors are believed to work on the exposed phospholipid surface of platelets, which helps to localise and accelerate these reactions.
23
Q
What role do calcium ions play?
A
Calcium ions play an important role in the binding of activated clotting factors to the phospholipid surfaces of platelets.
24
Q
What initiates coagulation?
A
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.
25
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.
26
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.
27
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.
28
What is plasma?
Simply, it is defined as the liquid component of the blood, comprising around 55% of a given blood volume.
29
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.
30
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.
31
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.
32
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.
33
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.
35
Which is the most abundant plasma protein?
Serum Albumin
36
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.
37
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.
38
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.
39
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.
40
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.
41
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.
42
Electrolytes make up what percentage of plasma?
They make up only 1%, but are major contributors of osmolarity.
43
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.
44
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.
45
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.
46
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.
47
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.
48
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.
49
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.
50
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
51
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.
52
Proteins make up what percentage of plasma?
Proteins make up around 7% of plasma and can be assayed by the technique of electrophoresis.
54
What is haemopoiesis?
The formation and development of blood cells.
54
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).
54
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.
55
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.
56
Outline the major function of erythrocytes
Their major function is oxygen transport and their approximate intramuscular lifespan is 120 days.
57
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.
58
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.
59
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).
60
Outline the major function of lymphocytes
Their main function is humoral and cell immunity. Their approximate intravascular lifespan varies greatly.
61
Outline the major function of platelets
Their main function is Haemostasis. Their approximate intravascular lifespan is 10 days.
62
How many blood cells does the average person produce each day?
500 Billion
63
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.
64
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.
65
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.
66
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.
67
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.
68
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.
69
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.
70
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.
71
How are megakaryoctopiesis and platelets produced?
Under the influence of thrombopoeitin.
72
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.
73
Outline the differentiation of myeloid progenitors
It produces erythroid, megakaryoctyes, platelets, granulocytes, neutrophils, basophils, acidophils and monocytes.
74
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.
75
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.
76
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.
77
Why is iron essential?
It is essential for health skin, mucous membranes, hair and nails.
78
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.
79
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.
80
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.
81
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.
82
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.
83
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.
84
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.
85
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.
86
What causes B12 deficiency?
1) Inadequate intake of folic acid (e.g. due to veganism).
2) Lack of acid in the stomach (achlorhydria)
3) Inadequate secretion of IF: pernicious anaemia (an autoimmune disorder)
4) Malabsorption (e.g. coeliac disease)
87
Outline the absorption of folic acid
Mainly in the small intestine, in the duodenum and jejunum. The total body stores last around 3 months and the recommended daily intake (RDA) is 100micrograms. Folate requirements increase surging pregnancy and in certain disease states with increased red cell production (e.g. sickle cell anaemia.
88
How are red cells destroyed?
At the end of their lifespan, red cells are phagocytosed by reticular endothelial macrophages in the spleen. They have their Haem moiety recycled. The iron is released from the Haem ring and bound to transferrin to be returned to the bone marrow to produce more red blood cells. The metabolism of Haem produces bilirubin, which is excreted in bile.
89
What are the 3 requirements of red blood cell function?
1) The integrity of the membrane
2) Haemoglobin structure and function
3) Cellular metabolism
A defect in any of these results in shortened erythrocyte survival (haemolysis)
90
Outline the structure of the red cell membrane
Erythrocytes are biconcave in shape, which helps their manoeuvrability through small blood vessels to deliver oxygen. The membrane is made up of a lipid belayer supported by protein cytoskeleton and contains transmembrane proteins. These maintain the integrity, shape and elasticity/deformability of the red cell.
91
What is hereditary spherocytosis?
This is an autosomal dominant gene, caused by the disruption of vertical linkages in membrane (usually ankyrin/spectrin). Spherocytes are red cells that are smaller, approximately spherical in shape and lack central pallor. They result from the loss of cell membrane without the loss of an equivalent amount of cytoplasm, so the cell is forced to round up. Red cells become less flexible and are forcefully removed by the spleen by haemolysis.
92
What is elliptocytosis?
Hereditary elliptocytosis is caused by the disruption of horizontal linkages in the membrane, these cells do not have an area of central pallor. Ellioptocytes may also occur in iron deficiency, resulting in a very pale colour, called a hyper chromic appearance.
93
Outline the function of Haemoglobin
The main function of erythrocytes is to carry oxygen. Oxygen is transported by the Haem moiety if Haemoglobin from the lungs to the tissues. Each erythrocytes contains ~300million Haemoglobin molecules.
94
Outline the structure of Haemoglobin
Adult Haemoglobin (Haemoglobin A) is a tetrameter: meaning that it is made up of 4 subunits, each composed of a globin chain (2 alpha and 2 beta) bound to a Haem group. Each Haem group consists of a ferrous iron ion (Fe2+) held in a ring known as a porphyrin. Each Fe2+ can bind to one oxygen molecule.
95
How does Haemoglobin differ at birth?
Fetal Haemoglobin (Haemoglobin F) has 2 alpha and 2 gamma globin chains. Haemoglobin A only becomes the prominent Haemoglobin at around 6 months of age. A very small amount of Haemoglobin F persists throughout adult life.
96
Outline the Oxygen Haemoglobin dissociation curve
The sigmoid oxygen Hb dissociation curve, facilitated oxygen delivery. It shows the the oxygen carrying capacity of Haemoglobin at different partial pressure of O2 (PO2). The sigmoid shape results from the interaction of bound oxygen molecules with incoming molecules. Cooperativity describes the binding of one oxygen molecule facilitating the second molecule binding. This is due to the induced conformational change in the structure of Haemoglobin by the binding of an oxygen molecule. As the Haemoglobin becomes saturated with oxygen, the curve levels out. P50 refers to the partial pressure of oxygen at which Haemoglobin is half saturated with oxygen.
97
What is the Bohr effect?
It refers it the observation that increases in the CO2 partial pressure of blood or decreases in blood pH, result in a lower affinity if Haemoglobin for oxygen. This leads to a rightward shift in the dissociation curve and yields enhanced unloading of oxygen by Haemoglobin and in metabolically active peripheral tissues such as exercising skeletal muscle. Increased skeletal muscle activity results in localised increases in the partial pressure of CO2 , which in turn reduces the local blood pH. This results in an enhanced unloading effect of bound oxygen by Haemoglobin passing through the metabolically active tissue and thus facilitates downloading of oxygen and its delivery to tissues. The Bohr effect enhances oxygen delivery proportionally to the metabolic activity of the tissue. Exercising skeletal muscles may also release lactic acidosis which further reduces the local blood pH and enhances the Bohr effect.
98
Outline the differing affinities for oxygen between Haemoglobin types
The oxygen affinity and therefore the position of the curve is also affected by the structure of Haemoglobin. Sickle Haemoglobin (Haemoglobin S) has a lower affinity for oxygen than Haemoglobin A, and thus has a rightward shift on the oxygen dissociation curve, meaning greater oxygen delivery to the tissues. Haemoglobin F, however, has a higher affinity for oxygen than Haemoglobin A, thereby favouring the take up of oxygen in the placenta. This high affinity for oxygen of Haemoglobin F is explained by its low affinity for 2,3 DPG (diphosphoglycerate) which is an organic phosphate, present in the red cells, that normally competes with oxygen for the binding of Haemoglobin.
99
Outline red cell metabolism
Metabolic pathways in the red cell are highly adapted to generate ATP and meet the energy requirements of the red cell. They are also important for Haemoglobin function and membrane integrity and deformability, as well as red blood cell volume.
100
What is 2,3-Diphosphoglycerate?
It is produced by the Rapaport-Leubering shuttle. It is an allosteric effector, meaning that it can modulate Haemoglobin oxygen affinity. It binds to the beta globin chain in the central cavity of the Haemoglobin chain. It plays a role in adaptive response to anaemia, hypoxia and high altitude.
101
What is Glucose-6-phosphate dehydrogenase (G6PD)?
It is an important enzyme in the hexose monophosphate (HMP) shunt, which is tightly coupled to Glutathione metabolism, which protects red cells from oxidant damage. Oxidants may be generated in the blood stream (e.g. during infection) or may be exogenous (e.g. drugs, broad beans). G6PD deficiency causes red cells right be vulnerable to oxidant damage.
102
Outline G6PD deficiency
It is the most prevalent enzyme disorder with more than 400 million sufferers worldwide. The gene for G6PD is found on the X chromosome so affected individuals are usually hemizygous males (but occasionally homozygous females). It usually causes intermittent, severe intravascular haemolysis (breakdown of unstable red cells in the blood vessel) as a result of infection or exposure to an exogenous oxidant. Extrinsic oxidants may be food stuffs (e.g. broad beans), chemicals or drugs. Its distribution parallels that of malaria, thus it confers a selective advantage with resistance to falciparum malaria.
103
Outline the link between G6PD and irregularly contracted cells
Episodes of intravascular haemolysis are associated with the appearance of considerable numbers of irregular contracted cells. These are irregular in outline but are smaller than normal cells and have lost their pallor. They usually result from oxidant damage to cell membrane and to the Haemoglobin. Haemoglobin is denatured and forms round inclusions known as Heinz bodies, which can be detected by a specific test.
104
Explain the names describing the size of red blood cells
1) Microcytic: red blood cells that are smaller than normal or an anaemia with small red blood cells. Caused by: a defect in Haem synthesis - iron deficiency or anaemia of chronic disease, or a defect in globin synthesis (thalassaemia) - defect in alpha chain synthesis or beta chain synthesis.
2) Normocytic: red blood cells that are of normal size or an anaemia with normal sized red blood cells.
3) Macrocytic: red blood cells that are larger than normal or an anaemia with large red blood cells. They can be of specific types: round, oval or polychromatic (blue tinge). Caused by: lack of Vitamin B12 or folic acid (megaloblastic anaemia), liver disease and ethanol toxicity, haemolysis (polychromasia) and pregnancy.
4) Anisocytosis: red cells showing more variation in size than is normal.
5) Poikilocytosis: red cells showing more variation in shape than is normal.
105
Explain the names describing the colour of red blood cells
1) Normal red cells have about a third of the diameter that is pale. This is a result of the disk shape of the red cement where the centre has less Haemoglobin and is therefore paler.
2) Hypochromia means that the cells have a larger area of central pallor than normal. This results from a lower Haemoglobin content and concentration and a flatter cell. Red cells that show hypochromia are hypochromic. Hypochromia and microcytosis often go hand in hand, due to iron deficiency and thalassaemia being common causes.
3) Polychromasia describe an increased blue tinge to the cytoplasm of a red cell. It indicates that the red cell is young. Polychromatic cells are larger than normal cells, as polychromasia is one of the causes of macrocytosis.
106
Outline the link between young red cells and reticulocytes
This allows young red cells to be detected by using a stain called new methylene blue for reticulocytes. This stains for their higher RNA content. Reticulocytosis refers to the presence of increased numbers of reticulocytes and may occur as a response to bleeding or haemolysis.
107
What are target cells?
They are red cells with an accumulation of Haemoglobin in the centre of the area of central pallor. They may occur in different conditions: obstructive jaundice, liver disease, haemoglobinopathies and hyposplenism (the spleen doesn’t function properly or has been removed).
108
What are sickle cells?
These are “sickle” shaped red cells. They result from the polymerisation of Haemoglobin S, which in the deoxygenated form is much less soluble than Haemoglobin A. Haemoglobin S occurs when one or two copies of an abnormal beta globin gene are inherited. The mutated beta Haemoglobin gene replaces a charged glutamic acid residue in position 6 with a uncharged valine molecule.
109
What are red cell fragments/schistocytes?
These are small piece of red cells. They indicate that a red cell has fragmented. This may result from a shearing process caused by the platelet rich blood clots in the small blood vessels (e.g. disseminated intravascular coagulation).
110
Outline normal and reference ranges
1) A reference range is derived from a carefully defined reference population. Samples are collected from healthy volunteers with defined characteristics. They are analysed using the instrument and techniques that will be used for patient samples. The data are analysed by an appropriate statistical technique. Not all results in the reference range are normal and not all outside the revenue range are abnormal.
2) A normal range is a much vaguer concept.
111
Outline the origin of white blood cells (leukocytes)
The multipotent stem cells gives rise to a myeloblast, which in turn can give rise to granulocytes and monocytes. Granulocytes refer to neutrophils, basophils and eosinophils, which have granules present in the cytoplasm that contain agents essential for their microbicidal function. Signalling through myeloid growth factors such as G-CSF, M-CSF and GM-CSF is essential for the proliferation and survival of myeloid cells.
112
Outline normal granulocyte maturation
Cell division occurs in myeloblasts, promyelocytes and myelocytes, but des not occur in metamyelocytes or band forms. Up to the neutrophil, all of these listed precursors are only present in the bone marrow.
113
What is the neutrophil?
The neutrophil granulocyte survives 7-10 hours in the circulation before migrating to tissues. The nucleus of the mature neutrophil is segmented (or lobulated). The first step in neutrophil migration to tissues is chemotaxis. Neutrophils become marginated in the vessel lumen, adhere to the endothelium and migrate into tissues.
114
Explain the function of neutrophil
Its main function is defence against infection; it phagocytoses and then kills microorganisms. Phagocytosis or microorganisms occurs following cytokines priming. Microorganisms are destroyed by the release of the neutrophils toxic intracellular contents by two mechanisms. The first is the superoxide dependent mechanism, whereby a reactive oxygen species is released, called a respiratory burst, which provides a substrate for the enzyme myeloproxidase (MPO), leading to the production of toxic acidic substances. The second mechanism is oxygen independent, a variety of Antimicrobial agents are released.
115
What is the eosinophil?
A myeloblast fan also give rise to eosinophil granulocytes. The eosinophil spends less time in circulation than the neutrophil does.
116
Explain the function of eosinophil
Its main function is defence against parasitic infection, and like neutrophils, it does this by phagocytosis. It also regulates some (immediate-type) hypersensitivity reactions.
117
What is the basophil?
A myeloblast can also give rise to a basophil granulocyte. It’s granules contain stores of histamine and heparin, as well as proteolytic enzymes.
118
Outline the functions of basophil
Basophils are involved in a variety of immune and inflammatory responses including:
1) Mediation of the immediate-type hypersensitivity reaction in which IgE-coated basophils release histamine and leukotrienes.
2) Modulation of inflammatory responses by releasing heparin and pro teases.
119
What is a monocyte?
The myeloid precursor gives rise to monocyte precursors and thence monocytes. Monocytes spend server alone days in circulation.
120
Outline the role of monocytes
Monocytes play several key roles that include phagocytosis and antigen presentation to lymphoid cells. Monocytes are the precursors of tissue macrophages and what circulates as monocytes, will migrate to tissues where they develop into macrophages (or histiocytes) and other specialised cells that have a phagocytic and scavenging function. Macrophages also store and release iron.
121
Outline the origin of lymphocytes
The multipotent haemopoietic stem cell also gives rise to a lymphoid stem cell. The lymphoid stem cell gives rise to T cells, B cells and natural killer (NK) cells. Lymphocytes recirculate the lymph nodes and other tissues and then back to the blood stream. Their intravascular lifespan is very variable.
122
What are B lymphocytes
These mature into plasma cells which produce antibodies.
123
What is humeral immunity?
Progenitor B cells develop sequentially in pre-B cells and mature B cells, during which there is gene rearrangement of the immunoglobulin heavy and light chains, leading to the production of surface immunoglobulins against many different antigens. The subsequent maturation of these cells requires exposure to antigens in the lymph nodes and other lymphoid tissue. This results in the recognition of non self antigens by the mature B cells and the production of specific immunity complements (antibodies).
124
What are plasma cells?
Plasma cells produce antibodies. They are characterised by their eccentric position, with their nucleus towards the edge of the cell and their vacuolated cytoplasm.
125
Outline the origin of T lymphocytes
Migration of lymphocyte progenitors from the lymphoid to the thymus, leads to the development of T lymphocytes. Gene rearrangement of the T cell receptors results in the expression of a large number of different T cell receptors, which are able to recognise a wide range of antigens. During their maturation, T cells acquire CD4 and CD8 cell surface markers. CD4+ lymphocytes are known as T Helper cells, and comprise the majority of the T cell population in the personal blood. CD8+ lymphocytes are cytotoxic T cells, and represent around 25% of circulating T cells. A small proportion of T cells in circulation are NK cells.
126
Outline the function of T lymphocytes
T lymphocytes are involved in cell mediated immunity.
1) NK cells are part of the innate immune system - they can kill tumour cells and virus infected cells.
2) T helper cells produce cytokines such as interferons, which activate the monocytes macrophage system and promote the synthesis of antibodies by B cells.
3) Cytotoxic T cells destroy the virus infected cells through binding of the T cell receptor.
127
Outline the appearance of lymphocytes
B and T cells cannot be readily distinguished by their morphological appearance on the blood film. Some lymphocytes are small and compact and that applies to both B and T cells. Other lymphocytes are lather with cytoplasmic granules and those are usually cytotoxic T cells or NK cells. If lymphocytes cannot be distinguished by their appearance, tests can be conduct that look for special features such as: the appearance of cell markers, Immuno globing or T cell receptors.
128
What is the most frequently used combination of stains?
H&E (Haematoxylin and Eosin) is one of the most frequently used combination of stains.
129
What is Haemotoxylin?
Haematoxylin is a purple-blue component that binds to acidic components of cells. In particular it binds DNA and shows up the nucleus. Many stains (including Leishman’s stain) contain a similar component that makes the nucleus visible.
130
What is Eosin?
Eosin is a pinkish stain that binds protein components, particularly in the cytoplasm. It is a bright red synthetic dye discovered in 1874 by Heinrich Caro, a German chemist. The German physician Paul Ehrlich published a technique for staining blood films in 1879 and coined the term 'eosinophil' to describe cells with granules which were readily stained by the dye.
131
What 5 factors affect the performance of a light microscope?
1) The wavelength of light.
2) The optical quality of lenses and all other components in the light path.
3) The refractive index of the medium through which the light passes (which is why the highest resolution lenses are designed to use oil instead of air between the objective lens and the slide).
3) The physical properties of the objective lens (property called the numerical aperture which is written on the lens next to its magnification, e.g. 10/0.25 means that lens has 10x magnification and a numerical aperture of 0.25. Hence, higher magnification does not necessarily mean higher resolution, though in fact it is normally the case that higher power lenses have better numerical apertures).
5) The geometry of the illuminating cone of light provided by the condenser lens (which depends in part on the design properties of the lens, but to a large degree on the way in which the condenser is focused and the iris diaphragms adjusted).
132
In practise, it is best to start with which power objective lens?
It is best to start with the low power (4x) objective lens, turned into position on the lens turret.
133
How is an image focused?
The main focusing knob may consist of an outer and inner part for coarse and fine focus, or there may be a single focusing knob which gives fine focus for the first quarter turn or so and then coarse focus engages.
134
How is the condenser focused?
Close the field iris and adjust the condenser focus knob until you get a sharp image of the edge of the disc of light coming through the iris diaphragm. This should be roughly in the centre of the field of view – if it is not, adjust it with the centering screws.
135
How is the field iris adjusted?
Open the field iris again and stop when the whole field of view is only just illuminated.
136
How is the condenser iris adjusted?
Start with the condenser iris fully open and then close it until it only just begins to darken the image. Examine the specimen, using the stage controls to move different regions of the slide into view and making fine adjustments of the focus as necessary. If you need to adjust the brightness of the image use the voltage control.
137
How do you change magnification?
Change magnification by swinging another objective lens into position and repeating the setting up procedure.
138
What do you do when having finished using the microscope?
Leave the lowest power objective in position, turn down the voltage control and and switch off the lamp.
139
How is a blood film prepared?
1) The blood sample is mixed throughly to ensure that all the cells are evenly distributed throughout the sample.
2) Once the sample is mixed, the blood dispenser is pushed through the lid of the sample, which allows a single drop to be taken from the sample (or take a drop of blood from a finger by using a sterile lancet to puncture the fleshy pad alongside the base of a fingernail) and placed onto the right hand side of the glass slide.
3) Another slide is taken to be used as a spreader. It is placed in front of the drop of blood and is pulled back gently, pushed forward and spread onto the rest of the slide. The direction of spreading should be so that the blood drop remains behind the spreader.
4) Once the slide has been made, label immediately to prevent misidentification. Label with the number from the sample and the name of the patient.
5) The blood should be left to dry before staining.
When preparing a blood film from uncoagulated blood it is important to work very quickly while the blood is still liquid.
140
What is Leishman’s stain?
Leishman’s stain contains a purple-blue dye, which stains nuclei, and a pink one staining components in the cytoplasm. Red cells look red, while white cells actually look blue due to the staining of the nucleus.
141
Outline the application of Leishman’s stain
1) ;The slide is first placed blood-side up in a rack over a draining dish.
2) 8 drops of Leishman’s stain are dropped on the using a dropper bottle so that the film is completely covered.
3) The slide is left for a minute to stain.
4) 8 drops of a buffer pH 6.8 (again from from a dropper bottle) should be dropped onto the slide and gentle rocking performed so that the buffer and stain are mixed. This is then left for 7 minutes.
5) The stain is poured off and the slide rinsed well with copious amounts of buffer, using a wash bottle.
6) The slide is dried by first tapping the edge of the slide on filter paper and then gently waving it in the air.
7) Once completely dry, the slide may be examined using the light microscope.
142
Outline the abnormalities can occur in leukocytes
```
Changes can be numerical, morphological, or both
Leukocytosis - too many white cells:
1) Neutrophilia
2) Eosinophilia
3) Basophilia
4) Lymphocytosis
5) Monocytosis
Leukopenia - reduction in total number of white cells:
1) Neutropenia
2) Lymphopenia
```
143
Which leukocyte most commonly causes leukocytosis and leukopenia?
They usually result from changes in the neutrophil numbers, as this is usually the most abundant leukocyte in the circulation.
144
Outline the causes of neutrophilia
It is most commonly caused by infection (particularly bacterial infection), inflammation, infarction or other tissue damage, myeloproliferative disorders (chronic myeloid leukaemia). Neutrophilia is also a common feature in pregnancy. Toxic granulation is a heavy flare granulation of neutrophils and may be also be feature of pregnancy. Neutrophilia may be seen following exercise (caused by a rapid shift of neutrophils from the marginated pool to the circulating pool) and after the administration of corticosteroids.
145
What may neutrophilia be accompanied by?
It may be accompanied by toxic changes and ‘left shift’. Left shift means that there is an increase in non-segmented neutrophils (band forms) or there are neutrophil precursors in the blood. It may also mean a presence of early myeloid cells such as metamyelocytes.
146
What is Chronic Myeloid Leukaemia (CML)?
There is an increase in all granulocytes - neutrophils, eosinophils and basophils - and their precursors, in both the blood and the bone marrow. CML results from a translocation (exchange of material) between chromosomes 9 and 22, occurring in a single haemopoietic stem cell. Philadelphia (Ph) chromosome is an abnormally short chromosome 22. Ph chromosome was the first consistent chromosome abnormality found in any malignancy. The gene provides the cell a growth and survival advantage and gives rise to a leukaemic clone.
147
How does the Ph chromosome give rise to a leukaemic clone?
ABL1 and BCR abnormal genes, are found on chromosomes 9 and 22 respectively. The ABL1 gene normally encodes a tyrosine kinase enzyme whose activities are tightly regulated. In the formation of the Ph translocation, 2 fusion genes are translocated. BCR-ABL1 on the Ph chromosome, 22, and ABL1-BCR, the reciprocal change on chromosome 9. BCR-ABL1 encodes a protein with uncontrolled tyrosine kinase activity, which gives rise to a leukaemic clone.
148
Outline the effects of CML
The spleen is enlarged (splenomegaly). The BCR-ABL1 protein signals between the cell surface and the nucleus. Growth factors attach to the receptors and tyrosine kinase signalling will then result in cell division.
149
Which inhibitors have revolutionised the treatment of CML?
The uncontrolled tyrosine kinase activity, resulting from the BCR-ABL1 fusion gene, can be inhibited by specific tyrosine kinase inhibitors leading to remission, and potentially cure of the disease.
150
Outline the causes of neutropenia
It can occur in a large number of conditions. This includes chemotherapy and radiotherapy. Neutropenia can also result from autoimmune disorders, sever bacterial infections, certain viral infections and drugs (e.g. some anticonvulsant and antipsychotic drugs and some antimalarials). Sometimes, neutropenia has a physiological basis (e.g, benign ethnic neutropenia in people of African or Afro-Caribbean ancestry). Patients with very low neutrophil counts (<0.5 x 10^9/l) are at a high risk of serious infection hand the need of urgent treatment with intravenous antibiotics.
151
What is a hypersegmented neutrophil?
A normal neutrophil should have between 3 and 5 segments or lobes. Neutrophil hypersegmentation means that there is an increase in the average number of neutrophil lobes or segments (“right shift”). It usually results from a lack of vitamin B12 or folic acid (megaloblastic anaemia), as the cell is able to grow but not divide.
152
Outline the causes of eosinophilia
Usually due to allergy or parasitic infection (e.g. strongyloidiasis, asthma, eczema or drug use). It can occur in leukaemia (e.g. CML). Eosinophils regulate immediate type hypersensitivity reactions, they inactive a histamine leukotrienes that are release by basophils and mast cells.
153
Outline the causes of basophilia
This is an uncommon finding that is usually due to leukaemia (e.g. CML) or a related condition. Other cells increased are myelocytes and band forms.
154
Outline the causes of monocytosis
Caused by infection (particularly chronic bacterial infection) or chronic inflammation. It is also caused by some forms of leukaemia.
155
Outline the causes of lymphocytosis
Often a response to a viral infection (transient), often “atypical lymphocytes”. Can also result from a lymphoproliferative disorder (persistent), such as lymphomas or chronic lymphomatic leaukaemias. Whooping cough (Bordatella pertussis) is an important cause of lymphocytosis in children.
156
Outline the characteristics of infectious mononucleosis
Key characteristics are the scalloped margins and the “hugging” of the surrounding red blood cells, as well as some lymphocytes having intensely basophilic cytoplasm.
157
What is chronic lymphocytic leukaemia (CLL)?
It has a characteristic squashed lymphocytes, known as “smear” or “smudge” cells. It is a lymphoproliferative disorder and the most common cause of persistent lymphocytosis in the elderly. Characterising the profile of cell surface markers expressed by lymphocytes, helps determine the cause of the lymphocytosis. This can be acgived using techniques such as flow cytometry.
158
What is the difference between acute and chronic lymphoid leukaemias?
Leukaemia is a cancer originating in haemopoietic or lymphoid cells.
1) In acute lymphoblastic leukaemia (ALL), there is an increase in very immature cells (lymphoblasts) with a failure of these to develop into mature lymphocytes. In ALL, the bone marrow is infiltrated by immature lymphoblasts, resulting in impaired haemopoiesis: lymphoblasts circulate in the peripheral blood. Acute conditions are sever and sudden in onset. If acute leukaemias are not treated, the disease is very aggressive and patients due quickly.
2) In chronic lymphoid (lymphocytic) leukaemias (CLL), the leukaemic cells are mature, although abnormal, T cells or B cells or NK cells. Chronic condition means that the disease and deterioration go in for a long period of time.
159
Outline the lymphoid differentiation of CLL
A mutation in a single haemopoietic lymphoid or myeloid stem cell in the bone marrow. Or in the case of CLL, there may be mutations of B or T lymphocytes, with more mature cells happening down the line.
160
Outline the lymphoid differentiation of ALL
Progenitors may a quite mutations which give result to acute leukaemias. So, mature B cells do not form and lymphoblasts fill the bone marrow, replacing the normal haemopoietic stem cells.
161
Outline the haematological features of ALL
Leukocytosis occurs with lymphoblasts in the blood. Suppression of normal haemopoiesis may result in anaemia (normocytic, normochromic), neutropenia and thrombocytopenia (low platelet count).
162
Outline cytogenetic and molecular genetic analysis
Cytogenetic/molecular genetic analysis is useful for managing the individual patient because it gives information about prognosis. These types of analysis advance knowledge of leukaemia as they have permitted the discovery of leukaemogenic mechanisms and the development of targeted treatment.
163
Outline the clinical features of ALL
Prominent bruising is caused by the reduced number of platelets (thrombocytopenia). Paleness is caused as a result of low Haemoglobin levels (anaemia). Both of these features are caused by the replacement if normal bone marrow cells with lymphoblasts, squeezing out normal haemopoietic activity.
164
Outline the treatment of ALL
It can be managed with supportive therapy in the form of red cell and platelet transfusions as well as antibiotics. Chemotherapy is given both systemically (through the body) and intrathecally (as the spinal cord relents a sanctuary site did the lymphoblasts).
165
Outline the causes of lymphopenia
It is defined as a total lymphocyte count <1 x 10^9/l. In normal blood, most lymphocytes are CD4+ T helper cells. The important causes of lymphopenia include HIV infection, chemotherapy, radiotherapy and corticosteroids. Patients with severe infection may develop a transient low lymphocyte count.
166
Outline the interpretation of white fells on a blood count and film
```
It is important to consider:
If there there is an abnormality
If so, then which cell line is abnormal
If there any clues in the clinical history of the patient
If there are any clues in the blood film
```
167
What is the function of tranexamic acid?
It reduces fibrinolytic activity, thereby reducing excessive bleeding. It is often used to treat excessive bleeding in major trauma.
168
Outline the importance of the ABO Blood group system
The group system is important as people have naturally occurring antibodies against any antigen NOT present on own red cells, from birth. The antibodies are IgM class, reactive at 37ºC and capable of fully activating complement, so are able to cause potentially fatal haemolysis (destruction of red cells) if incompatible blood is transfused. The blood group itself tells you which antigens are present on the red cell.
169
Outline the O blood group
The O blood group is the most frequent blood type in the UK, at 46%, and expresses no antigens. It can however, bind to anti-A and anti-B antibodies. Group O blood has no ABO antigens, so is safe to give to anyone in an emergency, until the patient’s own group is known.
170
Outline the A blood group
The A blood group is the second most frequent blood type in the UK, at 43%, and expresses the A antigen. It binds to the anti-A antibodies.
171
Outline the B blood group
The B blood group is the third most frequent blood type in the UK, at 8%, and expresses the B antigen. It binds to the anti-B antibodies.
172
Outline the AB blood group
The AB blood group is the least frequent blood type in the UK, at 3%, and expresses A and B antigens. It does not bind to any antibodies.
173
How are A and B antigens formed?
A and B antigens on red cells are formed by adding one or other sugar residue onto a common glycoprotein and fucose stem (H antigen) on the red cell membrane. N-acetyl galactosamine (galnac) is added to form an A antigen and galactose (gal) for a B antigen. Group O has neither A or B sugars, so consists of the H stem only.
174
What determines antigens on red cells?
Antigens are determined by corresponding genes. The A gene codes for an enzyme that adds N-acetyl galactosamine (galnac) to the common H antigen. The B gene codes for enzyme which adds galactose. A and B genes are co-dominant, O gene is recessive.
175
How can red cell incompatibility be shown in a lab?
In laboratory tests, IgM antibodies interact with corresponding antigens to cause agglutination (clumping), this shows red cell incompatibility, as this type of antigen antibody interaction can be fatal for patients.
176
Outline the Rh system
In this system, the most important antigen is D. The Blood groups are: RhD positive (if have D antigen is expressed), making up 85% of the population, or RhD negative (if D antigen is not expressed), 15% of the population. The ‘D’ gene codes for D antigen on red cell membrane, but the ‘d’ gene codes for no antigen and is recessive (no actual antigen). People who are RhD negative can make anti-D antibodies after they are exposed to the RhD antigen (sensitised) - either by transfusion of RhD positive blood, or in women if they are pregnant with an RhD positive foetus. Anti-D antibodies are IgG class antibodies.
177
Outline the 2 implications of anti-D antibodies after exposure to the D antigen
1) Transfusions: to avoid sensitisation, the patient must be given RhD negative blood, otherwise their anti-D antibodies would react with RhD positive blood, causing a delayed haemolytic transfusion reaction, with anaemia; high bilirubin (from breakdown of red cells), jaundice, etc.
2) HDN (haemolytic disease of the newborn): if a RhD negative mother has anti-D antibody, and her fetus is RhD positive, the mother’s IgG anti-D antibodies can cross the placenta (only IgG class antibodies can do this), where they can attach to the RhD positive red cells of the foetus and cause haemolysis of foetal red cells. In severe cases, this can lead to hydrops fetalis and even death. In less severe cases, the baby survives but after birth, the high bilirubin levels can cause brain damage or death. In this situation, the mother is given anti-D antibody so that the are the baby’s D antigen to cross the placenta to the mother, they’d be destroyed before the mother’s immune system can react.
178
Which blood type is given as emergency blood?
Group O negative blood (i.e. Rh D negative) is used as emergency blood when a patient’s blood group not known. Fresh Frozen Plasma (FFP) is given after massive bleeding to replace coagulation factors.
179
Outline the other antigens present on red cells
Dozens of other antigens are present on red cells. These are not routinely matched for (e.g. Rh group -C, c, E, e; others - Kell, Duffy, Kidd, etc). About 8% of patients transfused will form an antibody to one or more of these antigens. Once patients have formed an antibody, corresponding antigen negative blood is used; or else there is a risk of a delayed haemolytic reaction.
180
What is an antibody screen?
As well as testing ABO & RhD blood groups, in order to provide ABO and Rh compatible blood for a patient needing blood transfusion, an antibody screen on the patient’s plasma (known as 'group & screen'), is also necessary. It is performed to exclude any clinically significant immune antibodies.
181
Outline the steps of antibody screening
1) Patient plasma is incubated with 2 or 3 different fully typed 'screening' red cells, which are known to possess all the blood group antigens which matter clinically.
2) If the antibody screen is negative, any donor blood which is ABO & RhD compatible can be given.
3) If the antibody screen is positive, the antibody must be identified with the use of a large panel of red cells. Donor units of blood that lack the corresponding blood group antigen are then chosen for cross matching with the recipient's plasma prior to transfusion.
182
Outline the compatibility testing before transfusing patients
1) ABO group: test patient’s red cells with known anti-A and anti-B reagents
2) RhD group: test patient’s red cells with known anti-D and reagent
3) Select donor blood of the same ABO & RhD group.
4) Antibody screen +/- antibody panel, to identify antibody/ies.
5) Cross-match: patient’s serum mixed with chosen donor red cells - should not react: if reacts (agglutinates) = incompatible.
The compatibility test tests for: HIV, hepatitis (B,C and D), HTLV and syphilis. There is not test yet for vCJD.
183
Outline Prion disease and its issues regarding blood donation
Prion proteins have been found in membranes of lymphocytes and platelets and the prions of variant Creutzfeldt-Jacob disease (CJD) are found in lymphoreticular tissues. There have been 4 cases in the UK of variant CJD transmitted by transfusion of blood or blood products in humans, where donors who were entirely well, donated then years later developed vCJD. The last was around 10 years ago. A blood test to exclude any donor with vCJD is not yet available.
184
What is blood component therapy?
450ml blood is collected from a donor into a sterile plastic bag containing anti-coagulant. It is not efficient to use “whole blood” containing red cells, plasma & platelets – to transfuse patients, as most patients need only one of those components of blood (e.g. platelets, red cells, factor VIII, etc). Component therapy enables more efficient use of blood donations, and less waste of valuable resources. Also, when giving a patient a red cell transfusion for anaemia, they don’t need the excess fluid contained in the plasma part of whole blood: this just puts them at risk of fluid overload, if several units of red cells need to be transfused. Split one unit of blood by centrifuging whole bag (red cells bottom, platelets middle, plasma top) then squeeze each layer into satellite bags and cut free (closed system).
185
What are the 2 types of leukaemia?
As a cancer that arises as a result of mutation in a precursor of myeloid or lymphoid cells, leukaemias are therefore described as being myeloid or lymphoid. In this context, myeloid can include not only the precursors of granulocytes and monocytes but also cells of erythroid and megakaryocyte lineages.
186
How does leukaemia differ from many other cancers?
Leukaemia differs from many other cancers in that the abnormal cells circulate in the blood stream and migrate into various tissues. It is difficult to apply the concepts of local invasion and metastasis that are used to describe solid tumours to populations of cells that are normally mobile. The formation of localised tumour masses is also not inevitable in leukaemia, at least not in the earlier stages of the disease.
187
Outline the distinctions between acute and chronic leukaemia
An acute leukaemia is one that, if untreated, has profound pathological effects and leads to death in a matter of days, weeks or months. A chronic leukaemia is one that causes less impairment of function of normal tissues and, although it will eventually lead to death, this usually does not occur for a number of years. Leukaemias can therefore be acute (lymphoblastic or myeloid) or chronic (lymphocytic or myeloid).
188
How does leukaemia occur?
Leukaemia results from a number of mutations occurring in a primitive cell that, as a result, has a growth or survival advantage over normal cells that have not undergone mutation. That single cell gives rise to a clone that steadily replaces normal cells. The mutations concerned are in proto-oncogenes (also known as oncogenes) and sometimes also in tumour suppressor genes.
189
What causes mutations in somatic cells?
Mutation in a somatic cell may be the result of undetected exposure to mutagens or it may be a random, spontaneous process. The older a person is the more likely it is that enough spontaneous or induced mutations to have accumulated in a single cell for the cell to expand into a clone that replaces normal cells
190
In what ways can a leukaemic clone behave abnormally?
1) Growth that occurs without a dependence on growth factors
2) Continued proliferation without maturation
3) A failure to undergo normal cell death (apoptosis)
191
Outline the causes of each type of leukaemia
1) Acute lymphoblastic: usually unknown, sometimes mutagenic drugs or exposure to irradiation or chemicals in utero, possibly delayed exposure to a pathogen or pathogens. Most common form of leukaemia in infants.
2) Acute myeloid: usually unknown, sometimes irradiation, mutagenic drugs or chemicals (e.g. benzene, cigarette smoke).
3) Chronic lymphocytic (lymphoid): unknown but sometimes families are predisposed. Particularly effects the elderly.
4) Chronic myeloid: usually unknown, rarely irradiation or mutagenic drugs
192
What determines whether a leukaemia is chronic or acute?
It is the nature of the mutation that determines whether a leukaemia is acute or chronic. Acute leukaemias often result from mutations in genes encoding transcription factors with a resultant profound abnormality in the cells ability to mature. However the cells continue to proliferate so that there is an accumulation of primitive cells (blast cells) either lymphoblasts or myeloblasts. Whereas, in chronic myeloid leukaemia the mutation involves activation of signalling pathways within the cell (in CML this results from the fusion protein BCR-ABL1 encoded by the (9;22) Philadelphia chromosome). Cells can then proliferate without needing growth factors. Interaction with stroma may be abnormal and cell survival may be prolonged so that there is a steady expansion of the leukaemic clone. However maturation still occurs and, in the case of myeloid cells, mature end cells are still able to function. The impairment of normal physiological processes is therefore much less than in the acute leukaemias. The mutational events underlying chronic lymphocytic leukaemias are less well understood but they also result in the steady expansion of a clone of cells, in this case functionally useless; eventually there is impaired tissue function as the leukaemic clone replaces normal cells.
193
Outline the signs and symptoms of leukaemia
The clinical features may include:
1) Fatigue, lethargy, pallor (anaemia)
2) Fever and infections (neutropenia)
3) Bruising and petechiae (thrombocytopenia)
4) Bone pain (bone marrow expansion)
5) Abdominal enlargement (hepatomegaly, splenomegaly)
6) Lumps and swellings (lymphadenopathy)
194
What causes the symptoms of leukaemia?
The direct effects of the proliferation of the leukaemic cells, e.g. bone pain, enlarged liver (hepatomegaly), enlarged spleen (splenomegaly) and swollen lymph nodes (lymphadenopathy, mainly in lymphoid leukaemias)
2)The indirect effect of leukaemic cell proliferation, which leads to replacement of normal bone marrow cells by leukaemic cells (causing anaemia, thrombocytopenia, neutropenia).
195
How are the profile of the cell surface markers expressed characterised?
This can mean distinguishing between T and B lymphoid cells, andnis done using flow cytometry. A sample of bone marrow is taken for examination and cytogenetic/molecular analysis is performed on the blood and/or bone marrow to look for markers such as the Philadelphia chromosome (9;22). Cytogenetic/molecular genetic analysis is useful for managing the individual patient because it gives information about prognosis and also advances knowledge of leukaemia because it has permitted the discovery of leukaemogenic mechanisms and the development of targeted treatment.
196
What is anaemia?
Anaemia is a reduction in the haemoglobin concentration (Hb) in the circulating blood below what is normal for a healthy person of the same age and gender as the individual. Anaemia is usually associated with a reduction in the red blood cell count (RBC) and the haematocrit (Hct) (previously referred to as packed cell volume (PCV)). Anaemia is also caused when cytokines, such as TNF alpha and interleukins in chronic disease lead to a decrease in erythropoietin production and also prevent the normal flow of iron from the duodenum to the red blood cells.
197
Outline the 4 mechanisms that result in anaemia
1) Reduced production of red cells by the bone marrow
2) Loss of blood from the body
3) Reduced survival of red cells in the circulation (haemolysis)
4) Increased pooling of red cells in an enlarged spleen
198
Outline microcytic anaemia
In a microcytic anaemia red blood cells are small. They are referred to as microcytes. The size of red cells can be judged by looking at a blood film with a microscope or by measuring the mean cell volume (MCV) on an automated blood cell counter. Microcytic cells are usually also hypochromic, i.e. they appear pale when looked at with a microscope. The anaemia is therefore described as hypochromic and microcytic.
199
What causes microcytosis?
Microcytosis results from the reduced synthesis of haemoglobin. This can be caused by reduced synthesis of haem (iron deficiency or anaemia of chronic disease) or reduced synthesis of globin (thalassaemia).
200
What causes iron deficiency anaemia?
1) Blood loss: in particular gastrointestinal (which is often silent/asymptomatic) or in women, heavy menstrual bleeding (menorrhagia). This is the most common cause in adults, but hookworm is the most common cause worldwide.
2) Insufficient iron intake: either due to diet (e.g. vegetarianism) or malabsorption (e.g. coeliac disease - gluten induced or H. pylori gastritis.
3) Increased bodily requirements: may be due to physiology, pregnancy or infancy.
201
Outline the 3 stages of iron deficiency anaemia
1) Iron depletion: storage iron is reduced or absent
2) Iron deficiency: low serum iron and transferrin saturation
3) Iron deficiency anaemia: low Haemoglobin and Haematocrit
202
What causes anaemia of chronic disease (ACD)?
Anaemia of Chronic Disease (ACD) is anaemia in patients who are unwell; there is usually an inflammatory aspect to the underlying disease. Common causes include:
1) Rheumatoid arthritis
2) Autoimmune disease
3) Malignancy
4) Kidney disease
5) Infections such as TB or HIV
203
What are the clinical clues for anaemia of chronic disease (ACD)?
1) C-reactive protein is high (unlike iron deficiency)
2) Erythrocyte sedimentation rate (ESR) is high (unlike iron deficiency)
3) Ferritin is high
4) Transferrin is low
5) Acute phase proteins increase
204
What are the similarities between iron deficiency anaemia and anaemia of chronic disease?
In both:
1) Haemoglobin levels are low
2) The MCV is low
3) Serum iron levels are low
205
What are the differences between iron deficiency anaemia and anaemia of chronic disease?
1) Ferritin levels are low in iron deficiency, but high in ACD.
2) Transferrin levels are high in iron deficiency, but low in ACD.
206
What is macrocytic anaemia?
In a macrocytic anaemia red cells are larger than normal. They are referred to as macrocytes. The size of red cells can be assessed by examining a blood film or by noting an elevated MCV.
207
How does macrocytic anaemia occur?
Macrocytic anaemias usually result from abnormal haemopoiesis so that the red cell precursors continue to synthesise Haemoglobin and other cellular proteins, but fail to divide normally. As a result, the red cells end up larger than normal. One major cause of macrocytic anaemia is megaloblastic erythropoiesis, which refers to a delay in maturation of the nucleus while the cytoplasm continues to mature and the cell continues to grow. Megaloblasts are usually seen in the bone marrow, not the blood film.
208
What are the causes of megaloblastic anaemia?
1) Lack of vitamin B12 or folic acid
2) Use of drugs interfering with DNA synthesis
3) Liver disease and ethanol toxicity
4) Recent major blood loss with adequate iron stores (higher reticulocyte count)
5) Haemolytic anaemia (higher reticulocyte count)
209
What is normocytic anaemia?
In a normocytic anaemia the red cells are usually normally staining as well as normal in size, so the anaemia is referred to as normochromic normocytic. It is often caused by major blood loss, failure of production of red cells and pooling of red cells in the spleen.
210
What causes normocytic anaemia?
1) Gastrointestinal haemorrhage (trauma)
2) Early stage iron deficiency
3) Bone marrow failure or suppression (e.g. chemotherapy)
4) Bone marrow infiltration (e.g. leukaemia)
5) Hypersplenism (e.g. liver cirrhosis)
6) Splenic sequestration in sickle cell anaemia
211
Outline reticulocyte counting
The reticulocyte count involves exposing living red cells to a dye (new methylene blue) that stains the higher RNA content of young red blood cells so that they can be counted. An approximately equivalent observation on a routine blood film would be polychromasia. Polychromasia means that cells have a blue tinge, caused by the ribosomal RNA in young red cells, in addition to the pink colour of the haemoglobin
212
What do different reticulocyte counts show?
An increased reticulocyte count is seen as a response to haemolytic anaemia and recent blood loss and also as a response to treatment with iron, vitamin B12 or folic acid. A reduced reticulocyte count is seen when there is reduced output of red cells from the bone marrow.
213
Outline the regulation of the absorption of iron
Hepcidin works by binding to ferroportin and inducing its internalisation, thus preventing the efflux of iron from the duodenal enterocyte. The iron is then lost when the enterocyte dies and is shed into the gut lumen. When iron stores (ferritin) are full, there is upregulation of hepcidin expression and iron absorption is limited, whereas a requirement for increased erythropoeisis leads to a reduction in hepcidin and iron absorption is increased.
