Anil - Haematology by ICSM SU

Haematology MCD Year 2 Anil Chopra

Contents Contents.....................................................................................................................................................1 Haematology 1 - Iron deficiency...............................................................................................................2 Haematology 2 – B12 & Folic Acid Deficiency........................................................................................5 Haematology 3 – Haemoglobin and Thalassaemia....................................................................................8 Haematology 4 – Abnormal White Cell Counts......................................................................................11 Haematology 5 - Blood Diagnostic Parameters.......................................................................................15 Haematology 6 – Anaemia and Polycythaemia.......................................................................................18 Haematology 7 – Haemostasis.................................................................................................................22 Haematology 8 – Abnormalities of Haemostasis.....................................................................................26 Haematology 9 – Transfusion..................................................................................................................30

Haematology 1 - Iron deficiency Anil Chopra 1. Describe the role of iron in erythropoiesis. 2. List the dietary sources of iron, the factors influencing the absorption of iron, and the causes of iron deficiency. 3. Describe the clinical and haematological features of iron deficiency anaemia, and the diagnosis and management of iron deficiency. 4. Describe the clinical and haematological features of anaemia of chronic disease and explain how this is distinguished from iron deficiency.  IRON is mostly present in the body bound to haemoglobin Hb; therefore deficiencies of iron are primarily going to affect the haemoglobin and therefore the blood.  Each haem group is associated with a globin chain.

Erythropoiesis • • • • •

Iron binds to transferrin molecule Transferrin binds to transferrin receptor on the erythroblasts and passes over the iron molecules. Transferrin is released back into the circulation Erythroblasts undergo erythropoiesis (become red blood cell). Iron acts to increase the production of ferritin, and decrease the production of the transferrin receptor.

Sources of Iron include  Meat (haem)  Fish (haem)  Green Vegetables  Cereal, fortified Iron is absorbed in the duodenum.

FACTORS AFFECTING ABSORPTION - Increased…….ACID e.g. orange juice - Decreased……ALKALINE e.g. tea, chapattis Haem is better absorbed than free iron (up to 10% absorption) and its absorption is not adversely affected by other food components. In contrast, non-haem iron (i.e. Fe2+ and Fe3+) from vegetable sources are less well absorbed (1-2% absorption) and may be affected by other dietary factors.

Iron stores: (3-5 grams) - haemoglobin & myoglobin (2-3 grams) - ferritin and haemosiderin (1 gram) - plasma bound protein iron including transferrin (3 milligrams) Causes of iron deficiency  BLOOD LOSS (heavy periods, haemorrhage,)  DIET (vegans, vegetarians)  INCREASED NEED (pregnancy)  MALABSORBTION Classic iron deficiency: Hb MCV Serum iron Ferritin (useful!) Transferrin Transferrin saturation LOW

LOW LOW LOW LOW HIGH

Treatment is generally IRON REPLACEMENT usually by oral route (ferrous sulphate tablets) Anaemia of chronic disease is where there is no obvious cause apart from that the patient is unwell. It is normally associated with infection, inflammation or malignancy.

Classic anaemia of chronic disease: Hb LOW MCV LOW or N Serum iron LOW Ferritin HIGH or N Transferrin normal/low Transferrin saturation normal 1. RAISED C-reactive protein 2. RAISED Erythrocyte Sedimentation Rate 3. Acute phase response- INCREASES in - ferritin - FVIII - fibrinogen - immunoglobulins You can distinguish between anaemia of chronic disease and iron deficiency by using bone marrow aspirate. It is caused by cytokines (such as TNF alpha and interleukins) that are released at times of infection, inflammation or malignancy. They prevent the flow of iron into red blood cells which in turn blocks iron utilization by: 1. Stopping erythropoietin increase 2. Stopping iron flow out of cells 3. Increasing production of ferritin 4. Increasing death of red cells

Haematology 2 – B12 & Folic Acid Deficiency Anil Chopra 1.

Describe the role of vitamin B12 and folic acid in haemopoiesis, dietary sources and absorption of these vitamins, causes of deficiency, clinical and haematological features of vitamin B 12 and folic acid deficiency and the diagnosis, further investigation and management of these deficiencies Be able to explain that a. Synthesis of DNA requires both vitamin B12 and folate b. Integrity of the nervous system requires vitamin B12

B12  

Required for DNA synthesis Required for the integrity of the nervous system.

Folic acid  Required for DNA synthesis  Required for Homocystine metabolism Deficiency affects any cells which are rapidly dividing (bone marrow, epithelia of mouth and gut, embryos, gonads). Also leads to:     

Anaemia: weak, tired, short of breath Jaundice Glossitis and angular cheilosis Weight loss, change of bowel habit Sterility

Anaemia can either be MACROCYTIC or MEGALOBLASTIC Macrocytic Anaemia – generally defined by an increase in mean cell volume (MCV) This is usually measured by an automated full blood count. It can be caused by B12 or folate deficiency, liver disease, hypothyroidism, alcohol, drugs and haematological disorders. Megaloblastic Anaemia - abnormal red cell development.

Normal Red Cell Development 1) Proerythroblast: large cell with dark blue cytoplasm (high RNA content). It contains condensed chromatin. 2) Basophilic erythroblast: less RNA and more haemoglobin than proerythroblast. 3) Polychromatic Erythroblast: less RNA and more haemoglobin than basophilic erythroblast. 4) Pyknotic erythroblast: less RNA and more haemoglobin than polychromatic erythroblast. This moves out of the bone marrow. 5) Reticulocyte: nucleus is extruded completely and is found in the peripherals. 6) Mature Red blood Cells: formed from maturation of the reticulocytes. In megaloblasic anaemia, the maturation (disappearance) of the nucleus does not happen at the same time as the development of the cytoplasm. The nucleus or parts of it are therefore visible (these cells are known as megaloblasts). These therefore die in the bone marrow and so red cell production increases to compensate by a process known as incomplete erythropoiesis. White blood cells are also affected:  Hypersegmented neutrophils (nuclei have many segments)  Giant metamyelocytes (2-3 times their normal size) Can be defined by high MCV, although cells vary in size (anisocytosis) along with low haemoglobin, and low white blood cell count. Causes include B12 or folate deficiency or drugs. B12 and Folate Deficiencies Caused by: - inadequate intake - increased demand o pregnancy o adolescence o premature babies o cancers o anaemia - inadequate absorption o coeliac disease - excessive loss / utilisation Folate Deficiency Folate is contained in fresh leafy vegetables and animal products but is destroyed by overcooking, canning or processing. Diagnosis:  Full blood count (folate levels)  Blood film  Take history (look for alcoholism, skin disease, diet, illness, GI infection) Consequences  Megaloblastic, macrocytic anaemia  Neural tube defects in developing foetus o Spina bifida o Anencephaly  Increased risk of thrombosis in association with variant enzymes involved in homocysteine metabolism. Treatment  Folate replacement  All pregnant women take folic acid 0.4mg prior to conception and for the first 12 weeks B12 Deficiency

B12 is found in animal products so vegans are at risk. It is mainly caused not by inadequate intake, but by inadequate absorption. Normally, most B12 absorption is done by intrinsic factor which is synthesised in the stomach. B12-IF then binds to ileal receptors. Therefore low B12 can occur after a gastrectomy, gastric atrophy, or when the body produces antibodies to parietal cells and intrinsic factor. This autoimmune condition (known as Pernicious anaemia has a peak at around 60 years, is familial and increases the risk of stomach cancer in males). Another main cause of B12 malabsorption is small bowel disease. This can include Crohn’s or coeliac disease or bowel infections (tapeworm). Diagnosis  Measure vitamin B12 (will be low)  Neuro – examination will reveal absent reflexes.  Check levels of B12 intrinsic factor.  Antibodies for coeliac disease  Shillings test o Drink lots of radioactive B12 o Measure the excretion of B12 in the urine. o If there is no B12 in the urine repeat the test with intrinsic factor. Consequences Neurological problems  Bilateral peripheral neuropathy (loss of peripheral vision)  Subacute combined degeneration of the cord  Posterior and pyramidal tracts of the spinal cord are degraded  Optic atrophy (retinal tissue destroyed)  Dementia Symptoms  Paraesthesiae  Muscle weakness  Difficult walking  Visual impairment  Psychiatric disturbance

Haematology 3 – Haemoglobin and Thalassaemia Anil Chopra 1. Name the key components of haemoglobin 2. Name 3 types of haemoglobin 3. Describe the oxygen dissociation curve a) state what each axis represents b) explain the shape c) name 3 factors which affect it 4. Understand relationship between globin genes and different types of haemoglobin 5. Explain how genetic defects in globin genes lead to thalassaemia 6. Describe clinical and haematological features of thalassaemia major 7. Describe the principles of management of thalassaemia major 8. Describe the haematological and laboratory features of thalassaemia trait Hb is a composite protein as it contains both protein and Haem group & iron. (The deoxy version does not contain oxygen). There are different types of globin chain  α globin chains  β globin chain  γ globin chains  δ globin chains They always associate in pairs: α2 β2 = Hb A  95% α2 δ2 = Hb A2  1-3.5% α2 γ2 = Hb F  trace Each Hb molecules has 4 globin protein chains each with a haem group and a molecule of iron and each of which can potentially hold one O2 molecule. Haemoglobin Dissociation Curve Curve has SIGMOID shape 1. Haemoglobin can exist as a. deoxygenated (no oxygen) b. fully oxygenated (4 molecules) c. partially oxygenated (1,2,3) 2. Haemoglobin exists in 2 molecular configuration a. Tight – low affinity for O2 b. Relaxed – high affinity for O2

DEoxyhaemoglobin is in TIGHT configuration (counter-intuitive). This is because deoxyhaemoglobin is found in metabolically active tissues – it ensures that the tissues get the O2 rather than the haemoglobin. Deoxyhaemoglobin • No molecules of O2 • Low affinity for O2 • Tight “T” configuration Oxyhaemoglobin • 4 molecules of O2 • High affinity for O2 • Relaxed “R” configuration DeoxyHb is stabilised by: (i.e. reduces the affinity of deoxyhb even more) - H+ ions - CO2 (Bohr effect) - 2-3 DPG all theses shift the curve to the RIGHT. Globin Genes Organisation of Globin genes is complicated because: 1. Exception to the one gene=one protein hypothesis 2. Different globins occur at different times of life…embryo vs. fetus vs. adult 3. Genes occur in clusters…an alpha cluster and a beta cluster.

Exception for one gene – one protein theory is ALPHA GLOBIN. There are two alpha globin genes from each parent When forming an Hb molecule, you need a pair from the alpha cluster (on one chromosome) and a pair from the beta cluster (on another chromosome). There are a number of different combinations. Thalassaemia A disorder in which there is a reduction of the production of one of the globin chains. 5% of pop are carriers, but different gene defects occur in different parts of the world. Alpha thalassaemia • Caused by a gene deletion. • Usually found by co-incidence • occasional significant anaemia

• • •

Death in utero is rare Alpha globin chains are found in HbF so problems may start in utero There are two α genes in each cluster, so several syndromes are possible: • Alpha+ - ααα • Mild anaemia • MCV 77 • MCH 26 • •

Alphao - αα Mild anaemia • MCV 66 • MCH 24 (low)

• •

HbH - α Significant anaemia

• Hb Barts – no α chains! • Death in utero They need to be identified in pregnant women. Beta thalassaemia • Normally occurs as a point mutation • Co-incidental finding • Because there are no beta chains, the alpha chains form tetramers, α4 • These precipitate in bone marrow • They enter circulation and are removed in spleen • There are 2 types o If only one globin β chain allele bears a mutation, the disease is called β thalassemia minor (or sometimes called β thalassemia trait).  Hb may be normal  MCV low  MCH low  Red cell count increased  HbA2 increased [electrophoresis]  Normally does not affect patient too much o If both β chain alleles have thalassemia mutations, the disease is called β thalassemia major.  Severe life long anaemia  Fail to thrive  Malaise  Splenomegaly Treatment Blood transfusions are the main treatment however, because blood contains 200mg of iron per unit, it can lead to iron overload. Iron can accumulate in the liver (cirrhosis), heart (cardiac failure) and the endocrine glands. In order to prevent iron overload, patients have to be chelated with desferrioxamine (DFO). Patients dislike chelation as it is a subcutaneous infusing lasting for 8 hours a day. If they are not compliant and their ferritin levels are too high it can lead to arrhythmias. The other treatment is stem cell transplantation. Whilst this means that there is no need for transfusions, chelation and growth is normal, there is a risk with the transfusion, it causes infertility, and iron overload can still be a problem. On the whole it is generally a safe procedure.

Haematology 4 – Abnormal White Cell Counts Anil Chopra 1. 2. 3.

In a leucocytosis (increased white cell count) explain the importance of the differential count and peripheral blood morphology in planning further investigation. List the most common causes of an increased neutrophil, eosinophil and lymphocyte count. In a lymphocytosis, explain how to distinguish between a reactive polyclonal response to infection and a primary lymphoproliferative disorder (a monoclonal or malignant proliferation of lymphocytes such as chronic lymphocytic leukaemia)

Normal Full Blood Count Hb 12-16 g/dl Platelets 150-400 x 109/l WCC 4-11 x 109/l Neutrophils 2.5-7.5 x 109/l Monocytes 0.2-0.8 x 109/l Eosinophils 0.04-0.44 x 109/l Basophils 0.01-0.1 x 109/l Lymphocytes 1.5-3.5 x 109/l White blood cells originate in the bone marrow and proliferate from stem cells. Only when they are mature white cells can they pass into the peripheral blood. The differentiation is brought about by cytokines.

Conditions of Abnormal Cell Counts • • • • • •

Anaemia/polycythemia – deficiency/excess red blood cells Thrombo (cytopenia/cytosis) – deficiency/excess of platelets Neutro (penia/philia) – deficiency/excess of neutrophils Eosino (penia/philia) – deficiency/excess of eosinophils Leuco (penia/cytosis) – deficiency/excess of leukocytes Pancytopenia - all lineages reduced

Elevations in white blood cell count can be either reactive or primary: Primary Elevation of White Blood Cells:  Caused by Autonomous cell growth  Results in the formation of Immature/dysplastic/abnormal cells  Cell counts can be >10x normal  Often other counts often abnormal  Monoclonal – derived from single ancestor cell  Cause is normally underlying mutation Reactive Elevation of White blood cells:  Caused by increased cytokine production(appropriate or inappropriate)  Cells can show reactive changes  Cell counts are usually <5x normal  Often other cell counts are normal  Polyclonal – derived from different cells  There is no underlying mutation.

Measuring Raised Cell Counts • • • •

History and examination Haemoglobin and platelet count Automated differential Examine blood film

If an abnormality is found it is important to investigate whether other lineages are affected, including other white cell lineages, red cells and platelets. Also investigate whether it is only mature cells or immature cells that are also affected. Neutrophils • Present in Bone Marrow, blood and tissues • Life span 2-3 days in tissues (hours in PB) • 50% circulating neutrophils are marginated – stuck to walls of blood vessels as they pass into tissues and therefore are not counted in FBC. Neutrophilia Can develop in: • minutes > demargination • hours > early release from Bone Marrow • days > increased production (x3 in infection) – occurs in most systemic and local infections including viral, bacterial and fungal. Causes of Neutrophilia • Infection • Tissue inflammation (e.g. colitis, pancreatitis) • Underlying neoplasia • Myeloproliferative disorders • Physical stress, adrenaline, corticosteroids Chronic Myeloid Leukaemia:

Eosinophilia • Parasitic infestation • Allergic diseases e.g. asthma, rheumatoid, polyarteritis, pulmonary eosinophilia. • Neoplasms, esp. Hodgkin’s, T-cell NHL • Hypereosinophilic syndrome Monocytosis Monocytosis is rare but is seen in a number of chronic infections and primary haematological disorders • TB, brucella, typhoid • Viral; CMV, varicella zoster • Sarcoidosis • Chronic myelomonocytic leukaemia (MDS) Lymphocytosis Mature Cells: - reactive to infection, (EBV, CMV, Toxoplasma infectious hepatitis, rubella, herpes infections) chronic inflammation, underlying malignancy (sarcoidosis, neoplasia) o Reactive infections are more common in the elderly. o The morphology, immunophenotype and gene arrangement can normally distingusish between them. - caused by a primary disorder - can result in chronic lymphocytic leukaemia Immature Cells: - Caused by a primary disorder e.g. lymphoma, leukaemia - Acute lymphoblastic leukaemia Glandular Fever - Viral infection (Epstein Barr Virus) which invades B-lymphocytes via CD21 receptor - The infective B-cell proliferates and expresses the viral surface antigens. - Cytotoxic T-cell response clears the infection. In order to distinguish between the different types of Lymphocytosis: » Assess morphology of the cells o Atypical lymphocytes » Immunophenotype o Polyclonal: both kappa and lamda o Monoclonal: either kappa only or lambda only. » Gene re-arrangement o Southern Blot analysis

Haematology 5 - Blood Diagnostic Parameters Anil Chopra 1. 2. 3. 4.

Explain the origin, function and approximate intravascular life span of red cells, neutrophils and platelets Explain the function of monocytes, eosinophils and lymphocytes List the main physiological factors that influence the rate of red cell production. State the approximate intravascular life span of red cells, neutrophils and platelets.

All blood cells originate in the bone marrow and are derived from pluripotent haemopoietic stem cells. The pluripotent stem cells give rise to lymphoid stem cells and multipotent haemopoietic stem cells, from which red cells, granulocytes, monocytes and platelets are derived.

Pluripotent lymphoidmyeloid stem cell Multipotent myeloid stem cell

Granulocytemonocyte

Lymphoid stem cell

Megakaryocyte Erythroid

NK cell

T cell B cell

Red Blood Cells: the multipotent haemopoietic stem cells give rise to proerythroblasts, which give rise to erythroblasts which are converted into erythrocytes – process is called erythropoiesis and is regulated by erythropoietin a hormone made by the juxtatubular cells of the kidney in response to hypoxia (come also produced by hepatocytes). Red blood cells live for around 120 days and their function is to transport oxygen and carbon dioxide around the body. It is destroyed by phagocytic cells in the spleen. White Blood Cells Multipotent haemopoietic stem cells can also give rise to a myeloblast, which in turn can give rise to granulocytes and monocytes in the presence of cytokines such as G-CSF, M-CSF and GM-CSF. Neutrophils: these are present in the blood for around 7-10 hours before being transported into tissues. Its formation is as follows: Myeloblast Promyelocyte Myelocyte Band form  Neutrophil It phagocytoses invading microbes and then kills them in order to protect tissues. Eosinophils: these are present in the blood for even less time than neutrophils and their purpose is defence against parasitic pathogens. Basophils: also derived from myeloblasts – there are associated with allergic responses. Monocytes: monocytes are derived from haemopoietic stem cells which give rise to monocyte precursors and eventually monocytes. These spend several days in the circulation and their job is to

migrate to inflamed or infected tissue where they then become macrophages which phagocytose microbes and pathogens. They also store and release iron. Platelets: derived from haemopoietic stem cells which then give rise to megakaryocytes which in turn give rise to platelets. They survive in the circulation around 10 days. They promote blood coagulation in haemostasis. Lymphocytes: the lymphoid stem cell can give rise to B-cells, T-cells and NK-cells. These recirculate in the lymph nodes and round the blood. Their life span is very variable. Terminology  Anisocytosis – red cells show more variation in size than is normal  Poikilocytosis – red cells show more variation in shape than is normal  Microcytosis – red cells are smaller than normal  Macrocytosis – red cells are larger than normal  Microcyte – a red cell that is smaller than normal 

                        

Macrocyte – a red cell that is larger than normal. They can be of specific types • Round macrocytes • Oval macrocytes • Polychromatic macrocytes Microcytic – describes red cells that are smaller than normal or an anaemia with small red cells Normocytic – describes red cells that are of normal size or an anaemia with normal sized red cells Macrocytic – describes red cells that are larger than normal or an anaemia with large red cells Hypochromia – red blood cells that 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 described as hypochromic. Hypochromia and microcytosis often go together. Spherocytes - cells that are approximately spherical in shape. They therefore have a round, regular outline 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. Irregularly contracted cells - irregular in outline but are smaller than normal cells and have lost their central pallor. They usually result from oxidant damage to the cell membrane and to the haemoglobin. Polychromasia - describes an increased blue tinge to the cytoplasm of a red cell. It indicates that the red cell is young. Reticulocyte – immature red blood cell. These are recognised by staining with methylene blue which exposes a “reticulum”. Target cells - cells with an accumulation of haemoglobin in the centre of the area of central pallor. They occur in obstructive jaundice, liver disease, haemoglobinopathies and hyposplenism. Elliptocytes – red blood cells that are elliptical in shape. They occur in hereditary elliptocytosis and in iron deficiency. Sickle cells – red blood cells which are sickle or crescent shaped. They result from the polymerization of haemoglobin S when it is present in a high concentration. Fragments/ schistocytes - small pieces of red cells. They indicate that a red cell has fragmented. Rouleaux - stacks of red cells that resemble a pile of coins. They result from alterations in plasma proteins. Red cell agglutinates - differ from rouleaux in that they are irregular clumps, rather than tidy stacks. They usually result from antibody on the surface of the cells. A Howell-Jolly body - a nuclear remnant in a red cell. The commonest cause is lack of splenic function. Leucocytosis — too many white cells Leucopenia — too few white cells Neutrophilia — too many neutrophils Neutropenia — too few neutrophils Lymphocytosis — too many lymphocytes Eosinophilia — too many eosinophils Thrombocytosis – too many platelets Thrombocytopenia – too few platelets Erythrocytosis – too many red blood cells Reticulocytosis – too many reticulocytes

    

Lymphopenia – too few lymphocytes Atypical lymphocyte - an abnormal lymphocyte. Often the term is used to describe the abnormal cells present in infectious mononucleosis (‘glandular fever’). ‘Atypical mononuclear cell’ is an alternative term. Left shift - an increase in non-segmented neutrophils or that there are neutrophil precursors in the blood. Toxic granulation - heavy granulation of neutrophils. It results from infection, inflammation and tissue necrosis (but is also a normal feature of pregnancy). Hypersegmented neutrophil - there is an increase in the average number of neutrophil lobes or segments, usually resulting from a lack of vitamin B12 or folic acid.

Normal and Reference Ranges Full Blood Count Bear in mind that normal ranges may differ with age, gender, altitude, culture, diet, cigarette and alcohol intake.

Haematology 6 – Anaemia and Polycythaemia Anil Chopra 1. 2. 3. 4. 5.

Explain the term anaemia Describe the mechanisms underlying the development of anaemia Describe the classification of anaemia on the basis of red cell size List the common causes of microcytic, normocytic and macrocytic anaemia List causes of haemolytic anaemia and describe how you would recognise a haemolytic anaemia 6. Explain the possible mechanisms underlying polycythaemia Abbreviations used in the full blood count: • WBC – white blood cell count in a given volume of blood (x 109/l) • RBC – red blood cell count in a given volume of blood (x 1012/l) • Hb – haemoglobin concentration (g/dl or g/l) • PCV – packed cell volume (l/l) • Hct – haematocrit (same as packed cell volume) (l/l) • MCV – mean cell volume (fl) • MCH – mean cell haemoglobin (pg) • MCHC – mean cell haemoglobin concentration (g/l or g/dl) • Platelet count – the number of platelets in a given volume of blood (x 109/l)  The red and white blood cell counts are counted by large automated instruments that use electrical impulses generated by cells flowing through a light or electric field.  Haemoglobin is measured by spectrometry.  Packed cell volume or haematocrit is measured by centrifuging the blood.  Mean cell volume is calculated by dividing the total volume of red cells in a sample by the number of red cells in a sample (using machine).  The mean cell haemoglobin is the amount of haemoglobin in a given volume of blood divided by the number of red cells in the same volume.  The mean cell haemoglobin concentration is the amount of haemoglobin in a given volume of blood divided by the proportion of the sample represented by the red cells.  NB: the MCHC is the CONCENTRATION of haemoglobin per cell, whereas the MCH is just the AMOUNT.  Hypochromia correlates with MCHC. Anaemia Anaemia is defined as a reduction in the amount of haemoglobin in the blood below what is normal for that person’s age and gender. Characteristics of anaemia include: - Low Hb - Low RBC (red blood cell count) - Low PCV (packed cell volume/haemocrit) Mechanism of Anaemia • Reduced production of red cells/haemoglobin in the bone marrow • Loss of blood from the body • Reduced survival of red cells in the circulation • Pooling of red cells in a very large spleen

Causes of Anaemia - A condition resulting in the decrease of haem. E.g. reduced iron intake, cobalt poisoning. - A condition resulting in the decrease of globin. E.g. thalassaemia. There are 3 types of anaemia classified according to their cell size: Microcytic Anaemia (usually also hypochromic) » Causes o Defect in haem synthesis  Iron deficiency  Anaemia of chronic disease o Defect in globin synthesis (thalassaemia)  Defect in α chain synthesis (α thalassaemia)  Defect in β chain synthesis (β thalassaemia) Normocytic (usually also normochromic) » Causes o Recent blood loss o Failure of production of red cells o Pooling of red cells in the spleen o Peptic ulcer, oesophageal varices, trauma o Failure of production of red cells  Early stages of iron deficiency or anaemia of chronic disease  Renal failure  Bone marrow failure or suppression  Bone marrow infiltration o Hypersplenism, e.g. portal cirrhosis Macrocytic Anaemia (usually also normochromic) » Causes o Abnormal haemopoiesis o Red cell precursors continue to synthesize haemoglobin and other cellular proteins but they fail to divide normally o Red cells end up being larger than normal. o A particular type of abnormal haemopoiesis is megaloblastic erythropoiesis. This is where the maturation of the nucleus of red-blood cells is delayed whilst the cytoplasm grows at its normal rate. A megaloblast is therefore an abnormally large bone marrow erythroblast showing differentiation between nucleus and cytoplasm (not normal). o Premature release of cells from bone marrow. The MCV will be increased in this case because young red cells (reticulocytes) are larger. o These themselves have a number of causes including  Vitamin B12 and folic acid deficiency.  Use of drugs interfering with DNA synthesis.  Liver disease and toxicity.  Haemolytic anaemia –

Haemolytic Anaemia Haemolytic anaemia is defined by the shortened survival of red blood cells in the circulation either due to intrinsic red blood cell factors or extrinsic factors acting on red blood cells. It can be: • Inherited: can result from abnormalities in the cell membrane, the haemoglobin or the enzymes in the red cell • Acquired: usually results from extrinsic factors such as micro-organisms, chemicals or drugs that damage the red cell Extrinsic factors can interact with red cells that have an intrinsic abnormality. • Intravascular: occurs if there is very acute damage to red cells • Extravascular: occurs when defective red cells are removed by the spleen. (often it is both intravascular and extravascular). INHERITED ACQUIRED Diagnosis of Haemolytic Anaemia Abnormal red cell membrane, e.g. Patients will be suffering from hereditary haemolytic anaemia if: • There is an otherwise unexplained spherocytosis anaemia, which is Abnormal Hb, e.g. sickle cell anaemia normochromic and usually either normocytic or macrocytic Defect in glycolytic • There is evidence of morphologically abnormal red pathway, e.g. pyruvate kinase deficiency cells • There is evidence of increased red cell breakdown Defect in enzymes of pentose • There is evidence of shunt, e.g. G6PD deficiency increased bone marrow activity

Damage to red cell membrane, e.g AIHA or snake bite Damage to whole red cell, e.g, MAHA Oxidant exposure, damage to red cell membrane and Hb, e.g. dapsone or primaquine Precipitation of episodic haemolysis in individuals with enzyme deficiency

There are a number of different causes of haemolytic anaemia ranging from those which are genetically inherited e.g. sickle cell anaemia, spherocytosis, to those which are acquired e.g. malaria, autoimmune e.t.c. Hereditary Spherocytosis: this is caused by an inherited intrinsic defect in the cell membrane. They lose their membrane in the spleen and become spherocytic, and are then removed by extravascular haemolysis. The bone marrow responds to this by making more red blood cells resulting in reticulocytosis. The haemolysis results in increased bilirubin and hence jaundice. Treatment for hereditary spherocytosis includes: - Splenectomy (removal of spleen) – this is only done in extreme cases - Folic acid supplements.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency: this is an important enzyme in the protection of red blood cells from oxidant damage (oxidants may be generated in the blood stream from infections of endogenous products). The gene for this is on the X chromosome usually hemizygous males are affected. Haemolysis in G6PD deficiency is usually intravascular. It causes the appearance of irregularly contracted cells and the appearance of Heinz bodies. Autoimmune Haemolytic Anaemia: this results from the production f antibodies against red cell antigens. The immunoglobulin is bound to the red cell which results in macrophages removing parts of the membrane resulting in spherocytosis. Reticulocytes are also found (response from bone marrow) along with the detection of red cell antibodies and surface antigens on red cells. Treatment involves immunosuppression using corticosteroids or splenectomy in severe cases. Polycythaemia Refers to a large number of red cells in the blood. The Hb, RBC and PCV/Hct are all increased. It is normally due to an increase in the number of red blood cells – true polycythaemia (can occur because of a decrease in plasma volume – pseudopolycythaemia). It can occur because of transfusions (medical negligence), or as a result of high altitude (correctly). It can also occur in hypoxia, or when a renal tumour secretes erythropoietin, abnormal functioning of the bone marrow: - Inappropriately increased erythropoiesis that is independent, or largely independent, of erythropoietin - This condition is an intrinsic bone marrow disorder called polycythaemia vera - It is classified as a chronic myeloproliferative disorder. Complications include:  Hyperviscosity  Thick blood  Vascular obstruction Treatment  Removal of blood  Drugs to decrease bone marrow erythropoiesis.

Haematology 7 – Haemostasis Anil Chopra 1. 2. 3. 4.

Describe the normal haemostatic mechanisms including the interaction of vessel wall, platelets and clotting factors Describe the clinical features of bleeding due to thrombocytopenia and coagulation disorders, respectively Describe the use of laboratory tests to assess haemostasis Describe the principles of management of disorders of haemostasis

The response of endothelium to damage: 1. Vessel constriction This is mainly important in small blood vessels and is a local contractile response to injury, the precise mechanisms are uncertain. Functions of the Endothelium The endothelium maintains a barrier between blood and procoagulant subendothelial structures. It also synthesises PGI2, thrombomodulin, vWF, plasminogen activators

Smooth muscle cells

Composition:

vessel lumen

endothelial cells 2.

Formation of the Unstable Platelet Plug Bone Marrow

Synthesis of platelets

Circulation

basement membrane collagen, elastin, glycosaminoglycans; Maturation with granulation Proliferation Tissue factor 16 16 16

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Stem cell precursers, 2n, undergo nuclear replication to form megakaryocytes and become multinucleate

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Each megakaryocyte produces ~4000 platelets. Lifespan ~10 days, 1/3 stored in spleen

Surface glycoproteins:

Microtubules & actomyosin

GpIa GpIb GpIIb/IIIa

Glycogen

Structure of the Platelet:

Dense granules: ADP ATP Serotonin Ca2+

Phospholipid membrane Open cannalicular system

α granules:

growth factors fibrinogen factor V vWF

(Receptor for thrombin)

mitochondrion

Platelet Adhesion  Von Willebrand Factor is released  Platelets are bound to the sbasement membrane via glycoproteins Ia or Ib.  Thromoboxane is released resulting in… Platelet Aggregation  Glycoprotiens IIb and IIIa cause agglutination with intermediate fibrinogen and Ca2+ ions. In endothelial cells, prostacyclin PGI2 is synthesised from arachidonic acid. It inhibits platelet aggregation. In platelets, thromboxanes A2 is synthesised from arachidonic acid. It induces platelet aggregation. Tests for Platelet Aggregation Platelet Count: the normal range is between 150-400 x109/l. If it falls below 9 100 x 10 /l then bleeding can occur in trauma, if it falls below 40x109/l then bleeding can occur spontaneously, and if it falls below 10 x109/l, then spontaneous bleeding will be severe. Bleeding Time: a cut is made on the patients arm along with a cuff with 40mm/Hg pressure. Normal time is between 3-8 minutes. 3.

Stabilisation of the Plug With Fibrin

 Liver synthesises most coagulation proteins  Endothelial cells synthesise von Willebrand Factor.  Megakaryotes (platlets) synthesise factor V and von Willebrand factor. Platelets accelerate blood coagulation in a number of ways: » Factor IXa activates factor X (accelerated by factor VIIIa) » Factor X is converted to factor Xa » Factor Xa activates factor Va (accelerated by factor II) XII

XIIa

XIa

Not important for normal haemostasis

INTRINSIC PATHWAY

Prothrombin

EXTRINSIC PATHWAY

VIIa Ca 2

VIIIa Pl Ca 2+ X

Tissue factor (vessel damage)

VIIa Ca 2+

IXa

Blood coagulation

Va Pl Ca 2+

Fibrinogen

X COMMON PATHWAY

thrombin (IIa)

Fibrin thrombin XIIIa XIII Crosslinked

fibrin

Factor II causes thrombin generation

Warfarin Usage: anticoagulant. Used as a long-term anticoagulant. Mode of Action Warfarin inhibits the action of vitamin K in the liver. This results in the decreases in synthesis of most of the clotting factors needed in normal haemostasis. It also inhibits platelet aggregation, adhesion and the synthesis of thrombin. Heparin Usage: anticoagulant. Used in immediate anticoagulation in venous thrombosis and pulmonary embolism. Mode of Action Heparin accelerates the action of antithrombin – a plasma inhibitor. Test for Coagulation Activated partial thrombosis time – this detects abnormalities in the intrinsic and common pathways Prothrombin time - this detects abnormalities in the extrinsic and common pathways. Thrombin clotting time – detects abnormalities in fibrinogen conversion. » APTT and PT used together for screening for causes of bleeding disorders » APTT used to monitor heparin therapy in thrombosis » PT used for monitoring warfarin treatmentin thrombosis 4. Dissolution of Clot and Vessel Repair Fibrinolysis occurs in the presence of tPA – tissue plasminogen activator. This is where plasminogen is converted to plasmin. The plasmin then degrades the fibrin clot.

Haematology 8 – Abnormalities of Haemostasis Anil Chopra 1. 2. 3. 4.

Abnormalities Can Arise from: Lack of a specific factor: - Failure of production: congenital and acquired - Increased consumption/clearance Defective function of a factor - can be genetic or acquired (due to drugs, inhibition e.t.c.) Disorders of Primary Haemostasis 1. Disorders of Platelets: Thrombocytopenia Mechanisms of Thrombocytopenia  Failure of platelet production by megakaryocytes:  Generalised marrow failure e.g. aplastic anaemia, viruses, drugs, leukaemia, vitamin, B12/folate deficiency  Specific failure of megakaryocytes, e.g. myelodysplasia, congenital syndromes  Shortening the life (half-life) of platelets.  immune thrombocytopenic purpura (ITP) – antibodies which attack platelets and cause them to be phagocytoes by macrophages.  immune stimulated by drugs,  coagulation consumption  thrombotic thrombocytopenic purpura – causes multiple blood clots around the body idiopathically. Can be caused by a deficiency in the enzyme that breaks down von Willebrand factor  disseminated intravascular coagulation  Increasing the Activity of the Spleen in Pooling of platelets  Enlarged spleen – splenomegaly.  Also results in shorter half life of platelets Causes of Thrombocytopenia » Drugs o Aspirin and other NSAIDs – inhibit COX enzymes. o Clopidrogrel – ADP receptor inhibitor. » Congenital o Glycoprotein deficiencies such as Glanzman’s thrombasthenia (IIb/IIa), Bernard Soulier (GpIb) o Storage pool disease – lack of platelet granules. Symptoms and Signs of Thrombocytopenia » Typical primary haemostasis bleeding: o Prolonged bleeding from cuts o Epistaxes o Menorrhagia o Easy bruising o Prolonged bleeding after trauma or surgery » Petechiae (little red dots on skin from haemorrhage in blood vessels) – thrombocytopenia

2. Disorders of von willebrand factor: Von-Willebrand Disease Mechanisms of Von-Willebrand Disease Von Willebrand factor normally captures platelets by binding to the collagen attached to them, and by stabilising factor VIII. There are 3 types of hereditary von Willebrand disease, types 1 and 3 occur if there is a deficiency of vWF, and type 2 occurs if there is abnormal functioning. Symptoms and Signs of von Willebrand Disease  Prolonged bleeding from cuts  Epistaxes – nose bleeds  Menorrhagia – heavy bleeding from periods.  Easy bruising  Prolonged bleeding after trauma or surgery  Haemophilia-like bleeding in sever von Willebrand disease. 3. Disorder of the Vessel Wall There are a number of different vessels disorders that can cause bleeding: » Acquired o Scurvy – vitamin C deficiency o Steroid therapy o Aging (senile purpura) o Immune complex deposition o Allergic vasculitis – inflammation of blood vessels. » Inherited (rare) o Hereditary haemorrhagic telangiectasia – vascular deformations. o Ehlers-Danlos syndrome and other connective o tissue disorders, hypermobility Diagnosing Defects in Primary Haemostasis  Platelet count  Bleeding time  Assays of von Willebrand Factor  Clinical observation

Disorders of Secondary Haemostasis Normally the initial surge of thrombin whenever tissue is damaged triggers initiation. This will convert fibrinogen to fibrin and form the fibrin clot. If there are any factors that are deficient then there will be a thrombin deficienecy. Complications of Secondary Haemostatic Disorders • superficial cuts do not bleed (platelets) • bruising is common, nosebleeds are rare • spontaneous bleeding is deep, into muscles and joints • bleeding after trauma may be delayed and is prolonged • frequently restarts after stopping 1. Deficiency in Factor Production - Haemophilia Mechanisms of Haemophilia Haemophilia is an inherited condition:  Factor VIII deficiency leads to Haemophilia A  Factor IX deficiency leads to Haemophilia B Signs and Symptoms of Haemophilia  Deep bleeding (into joints) – haemarthrosis  Intramuscular injections should be avoided

Other Conditions Resulting in Secondary Haemostatic Disdorders  Acquired deficiency in factor production: o Liver disease o Drugs such as aspirin, warfarin  Consumption of coagulation factors o Immune diseases o Disseminated intravascular coagulation – blood coagulates around the body depleting the body of platlets. Diagnosing Defects in Primary Haemostasis - Screening tests o Prothrombin time o Activated partial thromboplastin time APTT - Factor Assays - Tests for inhibitors Disorders of Fibrinolysis  Disorders of fibrinolysis can cause abnormal bleeding but are rare  Hereditary – antiplasmin deficiency  Acquired – drugs such as tPA Genetics of Common Bleeding Disorders Von Willebrand Disease: autosomal dominant. Haemophilia: X-linked recessive Other diseases: autosomal recessive (therefore much less prevalent) Treatment of Haemostatic Disorderss Failure of Production  Factor Replacement Therapy: there are replacements available for all clotting factors except for factor V.  Platelet Replacement: platelet concentrates are given.  Stop Drugs: such as aspirin. Increased Destruction or Consumption  Treating Hypersensitvity: immunosuppression e.g. steroids  Splenectomy: removal of spleen  DDAVP: vasopressin – used to release endogenous stores of vWF and VIII. It is effective only in mild disorders.  Tranexamic Acid: inhibits fibrinolysis.

Haematology 9 – Transfusion Anil Chopra

1. 2. 3.

To be able to describe the major significant blood groups and their importance clinically To be able to describe the screening of blood donors undertaken and reasons why To be able to describe the various blood components used and the potential side effects of blood transfusion

Where is Blood From - Human source - 1 donor, 1 pint – max every 4 months - 10 000 units per day - Cannot be stockpiled as they are only kept for 5 weeks. Used only when benefits outweigh risks e.g. - massive bleeding - anaemia ABO Blood groups (most important) - Different sugars stuck on the end of a common fucose sugar stem. (either A, B, AB or O) - Group O has no sugar. - Group A has a N-acetyl galactosamine to common glycoprotein and fucose stem - Group B has galactose to common glycoprotein and fucose stem Genetics Genes that code for A and B are co-dominant whereas the gene that codes for type O is recessive.

People have antibodies (mainly IgM) against the antigens that are not present on their red blood cells, therefore if transfusion is incorrect it can be fatal: -

If O-type patient is given A, B or AB blood, then it can be fatal because patient will possess both anti-A and anti-B antibodies. They can only be given O blood. If A-type patient is given B or AB then it can be fatal because the patient possesses anti-B antibodies. They can be given O or A blood. If B-type patient is given A or AB then it can be fatal because the patient possesses anti-A antibodies. They can be given O or B blood. AB-type patients can be given any blood because they will have no antibodies.

Blood contains red blood cells plus plasma. In order to test for blood groups, blood is mixed with different known blood groups. If agglutination is seen (done by IgM antibodies) then the blood is incompatible. Rh Blood Groups Rhesus D is the most important blood group. Blood groups can be rhesus D + positive if they contain the rhesus D antigen, or rhesus D negative if they do not contain the antigen. Genetics - D allele codes for a rhesus antigen on red cell membranes - d allele codes for no antigen and is recessive. People who do not have the rhesus D antigen can synthesise it once they have been exposed to it in the first place. This is incredibly dangerous because, if they are sensitised and produce the D-antibody and they have a baby who is rhesus D negative, the antibodies will cross the placenta and kill/severely deform the baby. -

People who are Rh-D +ve can be given Rh-D +ve blood and Rh-D –ve blood. People who are Rh-D –ve blood can only be given Rh-D –ve blood.

There are other antigens present on other cells such as Rh-E, Rh-C which are less important and are not matched. They are however tested by an antibody screening. Blood Components

Normally 1 pint of blood is collected into a bag with anti-coagulant in it. The blood is then split up into its various components which may be needed by different patients at different times. This is done by centrifuging: Red Cells: we get one unit of red cells from 1 donor. This is stored at 4ºC and has a shelf life of approximately 5 weeks. These are given through a blood-giving set with a filter for debris. Occasionally they are frozen. Fresh Frozen Plasma: we also get 1 unit from one donor of around 300ml. It is frozen at -30ºC immediately in order to preserve coagulation factors. It has a shelf-life of 1 year and needs to be thawed for around 20-30 minutes before use. The dose is normally 12-15ml/kg = usually 2 or 3 units. This is normally used in coagulation factor disorders. Cryoprecipitate: this contains fibrinogen and factor VIII. It can also be stored at -30ºC for 1 year and 10 donors are needed for a single dose. It is used in massive bleeding if fibrinogen is very low. Platelets: we get one dose from 4 donors and they can be stored at 22ºC but only for 5 days. It is used in patents with low platlet counts, bone marrow disorders, cardiac bypass patients on anti-platelet drugs. Fractionated Products: these are fractionated and distilled like oil. These include:  Factor VIII and IX  For haemophilia A and B respectively (males)  Factor VIII for von Willebrand’s disease  Heat treated - viral inactivation  Recombinant factor VIII or IX alternatives increasingly used, but expensive  Immunoglobulins  Specific - tetanus; anti-D  Normal globulin - broad mix  IVIg - preop in patients with ITP or AIHA  Albimin  Burns  Kidney and liver dysfunction. Donors ⇒ Blood needs to be kept sage for recipient by testing for the following diseases: o Hepatitis B - HBsAg o Hepatitis C - anti-HCV o HIV - anti-HIV o HTLV - anti HTLV o Syphilis - TPHA (spirochete) o Some also tested for CMV (virus) ⇒ Donors need to be kept from harm so risky donors not used.