Haematology - Jack Roberts by ICSM SU

Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Haematology

MCD – Haematology Iron Deficiency 1. Describe the role of iron in erythropoiesis. • • • • •

Iron – essential component of many haem-containing molecules including enzymes, haemoglobin and myoglobin Iron → Haem + Globin → Haemoglobin (carries oxygen) Low iron = Low haemoglobin = anaemia Haem consists of a protoporhyrin ring with a ferrous iron (Fe 2+) at its centre Anaemia → Tissue Hypoxia → Increase in erythropoietin → red cell precursors (survive – grow – divide)

2. List the dietary sources of iron, the factors influencing the absorption of iron, and the causes of iron deficiency. • • • • • • • •

•

Red celles live for 120 days – to re-make huge numbers on daily basis, 20mg/day is required – most iron is recycled Loss – desquamation of gut/skin cell; bleeding – menstrual/pathological Men – 1mg/day; Women – 2mg/day o Diet provides 12-15mg/day – most not absorbed Sources – meat, offal and fish (haem iron), vegetables, whole grain cereals, chocolate Haem is better absorbed than free iron (up to 10% absorption) – not adversely affected by other food components Unable to absorb Fe3+; Fe2+ 1-2% o Improve – Acid pH, ascorbic acid, digestive enzymes o Inhibit – phytates and phosphates (tea) Systemic factors affecting absorption – iron deficiency, anaemia/hypoxia, pregnancy Total iron – 3-5g Metabolic pool Haemoglobin 2500 mg Myoglobin 500 mg Storage pool Ferritin and haemosiderin 0-1000 mg Transit pool Plasma protein-bound iron 3 mg e.g. transferrin bound Transferrin – holds to iron in circulation – glycoprotein made in the liver (2 iron binding sites) o

Total iron binding capacity (TIBC)

Interacts with a transferrin receptor on the surface of erythroblasts

The complex is then internalised, the iron removed, and then the transferrin re-circulated

Iron itself acts as a positive regulator of erythropoiesis and for the expression of the gene that codes for ferritin

Iron is a negative regulator for the expression of the gene that codes for the transferring receptor

3. Describe the clinical and haematological features of iron deficiency anaemia, and the diagnosis and management of iron deficiency. Hypochromatic microcytic anaemias – red cells have less haemoglobin than usual o Low MCH o Low MCHC (hypochromia) o Low MCV (small, microcytic) o IDA, ACD, Thalassaemias Iron deficient anaemia (IDA) – most common cause of anaemia worldwide o Blood loss - the main sources of blood loss are uterine in women of childbearing age group, followed by gastrointestinal blood loss, which may be overt or occult. o Dietary deficiency occurs in vegans and vegetarians with unbalanced diets poor in iron but can also occur in nonvegetarians. o Increased needs occur during childhood, especially during the pubertal growth spurt, and during child bearing. o Malabsorption is a less common cause of iron deficiency Clinical

Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Haematology o General – tiredness/lethargy, pale, short of breath (dyspnoea), palpitations o Specific – koilonychias (spoon shaped nails) Treatment – oral iron compounds (commonly ferrous sulphate). Side effects may include constipation and indigestion. In cases of difficulty, iron can be given parenterally (outside alimentary canal) – IM, IV

4. Describe the clinical and haematological features of anaemia of chronic disease and explain how this is distinguished from iron deficiency. No obvious cause except that the patient is ill Associated conditions – chronic infections (TB/HIV), chronic inflammation (RhA/SLE (lupus)), malignancy, miscellaneous e.g. cardiac failure Laboratory signs of being ill: o C-reactive protein (raised) o Erythrocyte Sedimentation Rate (raised) o Acute phase response- increases in ferritin; FVIII; fibrinogen; immunoglobulins Pathology – cytokines: o Stop erythropoietin increasing o Stop iron flowing out of cells o Increased production of ferritin o Increased death of red cells o Accumulation of excess iron in the bone marrow storage pool but with a block in iron incorporation into erythroblasts, which may lead to reduced haemoglobin synthesis and hypochromia Therefore - make less red cells; more red cells die; less availability of iron Cytokines include TNF alpha, interleukins The main pathological difference with iron deficient anaemia is the presence of raised marrow iron stores, a normal or raised serum ferritin and a normal to low serum transferrin/Total Iron Binding Capacity (TIBC)

5. Assessment of iron status – haematological features Parameter

Comment

Iron deficiency

ACD

Thalassaemia Trait

Non-specific screening

Low

Normal or low

MCV

Differential diagnosis of type of anaemia

Low (normal in early stages)

Low or normal

Low

Serum Iron

Low

Normal

TIBC (transferrin)

Raised

Normal or Low

Normal

Transferrin saturation

Low

Normal

Ferritin

Specific to IDA when low

Low

Normal or raised

Normal

Soluble TfR

Dependant on erythroid activity

High

Normal

Normal or slightly raised

BM iron stores

Gold standard for iron status

Absent

Increased

Normal or increased

Confirmation of thalassaemia trait – haemoglobin electrophoresis - additional type of haemoglobin is present Ferritin is not perfect – iron deficiency plus underlying chronic disease e.g. bleeding stomach ulcer (ferritin will be normal despite deficiency). Additional tests: o Blood film – may see changes associated with iron deficiency e.g. ellipocytes o Bone Marrow aspirate – slides can be stained to look for iron stores o Look for soluble transferring receptors Transferrin o IDA – increases o ACD – normal or even low o Therefore saturation will be low in IDA compared to normal in ACD

Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Haematology

Vitamin B12 and Folic Acid 1. Macrocytic anaemia Increase in MCV or the red cells (cells larger than normal) – measured by automated full blood count machine Causes – o Vitamin B12 or folate deficiency o Liver disease o Hypothyroidism o Excessive alcohol consumption o Drugs e.g. azathioprine, zidovudine o Haematological disorders  myelodysplasia  aplastic anaemia  reticulocytosis e.g. chronic haemolytic anaemias

2. Megaloblastic anaemia Abnormal but distinct morphological appearance of early and developing blood cells – discernable by light microscopy Normal:

Proerythroblast – dark blue cytoplasm – high RNA content; nucleus – slightly condensed chromatin Erythroblast – progressively less RNA, more haemoglobin Late erythroblast – pink rather that blue cytoplasm – confined to bone marrow Nuclear chromatin becomes more condensed until extruded completely → reticulocyte – may be found in peripheral blood – precursors of RBC Megaloblastic anaemia: o Megaloblast (vs. normoblast) o Anisocytosis o Large red cells (MCV high) o Hypersegmented neutrophils - >5 abnormal o Giant metamyelocytes o WBC and platelet count may also be low Delayed maturation of the nuclei – many blood cells die in the bone marrow – R cell production increases to compensate – ineffective erythropoiesis

3. Vitamin B12 Deficiency Haematinic deficiencies – consider intake; demand; absorption, loss or utilisation Inadequate intake is rare o Vitamin B12 is found in animal products, so vegans are at risk o Abnormal bacterial flora in the small bowel (e.g. associated with stagnant loops) can consume vitamin B 12 Increased demands are usually readily covered by the vitamin B12 stores, which are relatively large in relation to daily needs and usually sufficient to last for many years. Absorption of B12 is complicated and failure of absorption is the commonest cause of B12 deficiency o B12 is absorbed in the small bowel following combination with intrinsic factor. Intrinsic factor is made in the stomach (parietal cells). o B12 absorption may be impaired in the following situations:  reduction in active intrinsic factor  post gastrectomy  autoimmune gastric atrophy (“pernicious anaemia”)  small bowel disease  surgical resection  Crohn’s disease  coeliac disease Excessive losses: this is not a common cause of B12 deficiency

Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Haematology Consequences of vitamin B12 deficiency: o Megaloblastic anaemia o Neurological problems:  peripheral neuropathy  sub-acute combined degeneration of the spinal cord  optic neuropathy  dementia Laboratory diagnosis of B12 deficiency: o Blood count and film o Serum B12 level o Shilling test may be necessary to determine the cause of the deficiency.  Radiolabelled B12 is given orally and its excretion in the urine is measured, after first having saturated the serum B12-binding proteins by giving an intramuscular injection of non-radio-active B 12.  Any radiolabelled B12 detected in the urine must have been successfully absorbed in the small intestine.  If the excretion is low then the test is repeated with the addition of intrinsic factor.  If this restores the excretion of B12 to normal it is possible to conclude that the defect lies with a lack of intrinsic factor secretion.  The detection of anti-parietal cell and anti-intrinsic factor antibodies in the blood, particularly the latter, would be additional evidence that a patient had pernicious anaemia.

4. Folate Deficiency Inadequate intake is common – through either ignorance, poverty or apathy Folate is found in animal and plant products but is readily destroyed by cooking, canning and processing Daily requirements = 100µg, store = 10mg (3-4 months) Common in elderly and alcoholics Increased demand is also a common cause of deficiency: o Physiological – pregnancy, lactation, adolescence, premature babies o Pathological – an excessive turnover on cells as may occur with haemolytic anaemias, malignancy or erythroderma Absorption occurs in the duodenum and jejunum Failure of absorption is rare unless these is widespread disease of the small bowel such as in coeliac disease Excessive loss is not a normal cause of folate deficiency Consequences of deficiency: o Megaloblastic anaemia o Neural tube defects in developing fetus – spina bifida, anencephaly o A possible chance of increased risk of coronary artery disease if associated with variant enzymes in the folate metabolic pathway o Required for homocysteine metabolism – high levels of homocysteine is associated with atherosclerosis and premature vascular disease Lab Diagnosis: o Full Blood Count and film o Serum folate o Red cell folate – gives a better indication of body stores of folate whereas serum folate reflects recent intake

5. Explain that synthesis of DNA requires both vitamin B12 and folate Both are required for the metabolic pathway that ultimately ends in DNA synthesis DNA requires folic acid in the tetrahydrofolate (FH4) form to act as a co-factor in synthesis, therefore folic acid deficiency hinders this process. Vitamin B12 is required to produce FH4 and so indirectly affects DNA synthesis. All cells which are dividing are thus affected, but the actively proliferating bone marrow cells (i.e. where haemopoiesis occurs) are particularly affected. The slowdown of DNA synthesis and the prolonged cell cycling (of cells already in the circulation) causes the discharge of blood cells before they have undergone the full number of divisions (i.e. premature blood cells are released into the circulation). Consequently, these prematurely released cells tend to be enlarged (red blood cells) – this leads to anaemia that is both macrocytic and megaloblastic.

Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Haematology

The Haemoglobin Molecule and Thalassaemia 1. Describe the structure and function of the haemoglobin molecule and list the normal haemoglobins in the fetal, neonatal and adult periods. Each haemoglobin molecule contains: o 4 globin protein chains (two pairs 2+2) o 4 haem groups (protoporphyrin rings) o 4 molecules of iron o Up to 4 molecules of oxygen The main function of Hb is the carriage of oxygen from the lungs to the tissues. Hb molecules can exist in two spatial configurations: o Deoxy haemoglobin exists in a tight (T) configuration and has a relatively low affinity for oxygen. o Oxygen molecules are taken up sequentially by the 4 haem groups and at some point the partially liganded Hb molecule switches. o Relaxed (R) configuration has a markedly higher affinity for oxygen. This can be represented diagrammatically by the oxygen dissociation curve. o H+ ions, CO2 and 2,3-DPG (an organic phosphate compound) all stabilise the T form of the oxygen molecule by forming H bonds and thus decrease the oxygen affinity of the molecule. o This is represented on the oxygen dissociation curve as a shift to the right i.e a higher concentration of O2 is needed for maximum O2 saturation if the concentration of CO2, H+ ions or 2,3-DPG are high. o Thus in metabolically active tissues where the concentration of H ions and CO2 are high, oxyhaemoglobin will assume the T configuration and give up oxygen readily. o Conversely in the lungs where CO2 is exhaled, oxygen affinity is higher. This effect of CO2 on the affinity of Hb for oxygen is called the Bohr effect. Six different types of globin exist, three of which are transient embryonic that code for the globins are located in two clusters.

Adult Foetal Embryonic

Alpha Cluster – Chromosome 16 α ζ

haemoglobins. The genes

Beta Cluster Chromosome 11 β,δ γ ε

There are two alpha genes for the alpha globin protein. Two are inherited from each parent so adults have four alpha globin genes in total. Foetal haemoglobin – predominantly Hb F (α 2γ2) Adult haemoglobin o >95% Hb A (α2β2) o 1-3.5% Hb A2 (α2δ2) o Trace Hb F (α2γ2) If someone has a reduction in the number of beta chains (e.g. thalassaemia trait) then HbA2 would be proportionately increased and detected as a higher percentage on haemoglobin electrophoresis.

Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Haematology (δ2γ 2) and (β2δ2) do not exist

2. Describe the genes controlling haemoglobin synthesis and explain how genetic defects lead to α and β thalassaemias. Thalassaemias are disorders in which there is reduced production of one of the two types of globin chains in haemoglobin leading to imbalanced globin chain synthesis 5% of the world population estimated to be carriers Underproduction of a globin chain may be the result of o Gene missing completely (deletion) o Gene abnormal  start signal; no transcription  mRNA unstable; no translation  protein abnormal/dysfunctional

3. Describe briefly the clinical and haematological features of α thalassaemia Alpha chains are found in HbA and HbF so alpha thalassaemia may present clinically in utero Alpha thalassaemia is usually (>80% cases) due to a deletion of one or more alpha genes Each alpha cluster (one on each chromosome) has two alpha genes - four syndromes are possible, each with an increasing degree of anaemia and associated morbidity o α+ trait (ααα) Mild anaemia o α0 trait (αα) Mild anaemia o Hb H disease (α) Significant anaemia o Hb Bart’s hydrops fetalis Death in utero Genetic screening – all women screened for [--αα] → partner [--αα] Could give baby of [-- --] α+ trait common in Africa and α0 trait particularly common in SE Asia.

4. Describe briefly the clinical and haematological features of β thalassaemia major and the principles of management. Most types of β thalassaemia are due to point mutations and over 100 different mutations have been described Severe defect in BOTH beta chains o no problems in utero because HbF is alpha and gamma chains o at 2-3 months, become profoundly anaemic with Hb 3 or 4g/dl o failure to thrive and general malaise In the absence of beta chains, alpha chains accumulate and precipitate in the bone marrow causing cell death; this is called ineffective erythropoiesis Cells which do manage to mature and enter the circulation contain β-chain inclusions and are removed by the spleen which subsequently enlarges The anaemia stimulates erythropoietin production and this causes expansion of the bone marrow in the skull and long bones A patient with thalassaemia major has profound anaemia and requires regular blood transfusions to survive A patient with thalassaemia intermedia, has anaemia but does not require regular blood transfusions No transfusions - die aged 7 Blood transfusion - die aged 25 from iron overload or viral transmission e.g. hepatitis B and C and HIV Each unit of blood contains 200mg iron and this accumulates in the liver, heart and endocrine glands - the effects of this start to appear by the end of the first decade o Secondary sexual development may be delayed or absent o hypoparathyroidism and adrenal insufficiency may become apparent o progressive liver and cardiac damage occur and liver damage from the iron overload may be exacerbated further by infectious hepatitis Removal of iron is difficult. Currently the most successful drug is an iron-chelating agent called desferrioxamine o Not orally active o Subcutaneous infusion o Expensive o 90% get into 30’s if well chelated – death usually (60%) a result of cardiac failure 2° to iron overload Bone marrow transplantation has the potential to cure thalassaemia major and should be considered in transfusion-dependent thalassaemics under the age of 16 years who have an HLA-identical sibling greater than 18 months of age

5. Describe the haematological features of β thalassaemia trait, how it is diagnosed and why this is important. Carrier state for abnormal β globin gene, usually clinically silent o Hb may be normal

Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Haematology o MCV low (microcytosis) o MCH low o Normal MCHC o Red cell count increased o HbA2 increased [electrophoresis] There are two situations in which identifying patients as having β thalassaemia trait (DNA analysis) is of value: o Microcytosis may be misinterpreted as iron deficiency if the raised red cell count and normal MCHC are not noted If these patients are then put on long term iron they can become iron overloaded o It is important to identify pregnant patients with thalassaemia trait so that their partners can be tested and the couple can be counselled about their chance of having a baby with clinically significant thalassaemia and can be offered further testing.

6. Describe how β thalassaemia trait can be differentiated from iron deficiency anaemia and the anaemia of chronic disease. See previous table

Abnormal White Cell Counts 1. In a leucocytosis (increased white cell count) explain the importance of the differential count and peripheral blood morphology in planning further investigation. White cells consist of two main groups which are present throughout body tissues and play a central role in the response to infection mediated via phagocytosis and soluble proteins of the immunoglobulin and complement system: Phagocytes o Monocytes o Granulocytes → neutrophils, basophils and eosinophils Immunocytes o T and B lymphocytes and NK cells Differentiation and maturation: Myeloblast → Promyelocyte → Myelocyte → Metamyelocyte → Neutrophils present in the peripheral blood

Bone marrow is the principal source of WBC’s where they proliferate and differentiate from common stem cells WBC’s mature in the peripheral blood Cytokines influence differentiation and proliferation, processes which are directed by DNA (damage to which leads to cancer →leukaemia - lymphoma/myeloma) Lymphoid cell proliferation is governed by IL2 Myeloid differentiation is governed by G-CSF and M-CSF Erythroid differentiation is governed by erythropoietin When an elevated WBC count is identified it is necessary to first look at the automated differential Is the leukocytosis due to elevated numbers of a particular cell type such as in lymphocytosis, neutrophilia or eosinophilia or alternatively due to an increase in all cell types A blood film will determine whether only mature cells are present in the peripheral blood, or whether immature forms such as myeloblasts or lymphoblasts are present The morphology of white cells will also identify other reactive changes such as toxic granulation of neutrophils By looking at elevate WBC’s in this fashion allows one to identify the type of underling problem

Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Haematology

2. List the most common causes of an increased neutrophil, eosinophil and lymphocyte count. Neutrophilia Neutrophils are present in the BM, blood and tissues. Their life span in the tissue is around 2-3 days. 50% of neutrophils are marginated in the tissues and so are not counted on a full blood count Neutrophilia can develop in: o Minutes due to demargination o Hours due to early release from the BM o Days due to increased production (x3 during infection) Neutrophilia is defined as an absolute neutrophils count of more than 7.5x109/L in adults Common causes include: Bacterial Infection – commonest is an acute bacterial infection such as chest, or urinary tract. Count is raised and may show toxic granulation with the presence of increased numbers on cytoplasmic granules and vacuoles. Some infections such as typhoid and viral infections do not lead to raised neutrophil counts Inflammation and tissue necrosis – such as in appendicitis Underlying neoplastic disease such as carcinoma or lymphoma may produce reactive neutrophilia due to increased production of cytokines Myeloproliferative disorders such as chronic myeloid leukaemia (CML). Less mature forms such as myelocytes and myeloblasts are present Demargination – neutrophils within the blood stream are divided between the circulating and the marginated pool. Physical exercise and acute, severe physical stress can increase the circulating neutrophils count by moving neutrophils from the endothelial surface of small blood vessels into the flowing blood. Corticosteroids also raise the neutrophil count Eosinophilia: Defined as a count of more than 0.4x109/L Most common causes are: o Parasite infestations such as filarasis o Allergic disease such as asthma and eczema Hodgkin’s disease – a cancer of the lymphatic system which may produce a reactive eosinophila Idiopathic hypereosinophilic syndrome Monocytosis: Is uncommon but may be seen in some chronic bacterial infections that do not produce a neutrophils response such as TB and typhoid Also occurs in primary haematological conditions such as chronic myelomonocytic leukaemia Lymphocytosis: Defined as a lymphocyte count of more than 4.0x109/L in adults Many causes which can be divided into two groups: o Primary lymphocytosis – a malignant clonal proliferation of lymphocytes such as in lymphocytic leukaemia or

Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Haematology lymphoma Secondary reactive lymphocytosis – a polyclonal reactive proliferation as a result of infection or inflammation  Infections - EBV, CMV, Rubella, Hepatitis and TB  Autoimmune disorders When lymphocytosis is identified a blood film must be examined for: o Atypical/reactive lymphocytes seen in mononucleosis syndromes o Immediate response to acute stress such as in MI o Small lymphocytes and smudge cells seen in chronic lymphocytic leukaemia o Primitive blasts seen in acute lymphoblastic leukaemia o

3. 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). A full blood count may reveal the presence of an increased lymphocyte count. This can broadly be considered to be due to either a neoplastic proliferation of lymphocytes (a form of lymphoma or lymphoid leukaemia) or, alternatively, it may be a reaction to an underlying disorder such as a viral infection, for example ‘glandular fever’ (infectious mononucleosis). The approach to diagnosing the cause of a lymphocytosis would consider the age, clinical features and laboratory investigation. In the laboratory, morphology may simply reveal mature lymphocytes. The presence of abnormal forms such as smear cell (lymphocytes damaged by blood film preparation) or blast cells are suggestive of a lymphoproliferative disorder such as leukaemia or lymphoma. If required, further laboratory tests can distinguish between monoclonal (primary) and polyclonal (reactive/secondary) lymphocytes. Individual B-lymphocytes express either κ or λ light chains on the cell surface. In a population of monoclonal B cells only one immunoglobulin light chain type, either κ or λ will be present whereas in a reactive increase in B cells there will be a mixed population of κ and λ expressing cells. A more demanding assay using the T cell receptor genes can be used to study the rarer finding of a T cell lymphocytosis.

Blood Diagnostic Parameters, Terminology, and Reference Ranges 1. State the function and life span of the following blood cell types: red cells, neutrophils, platelets, monocytes, and eosinophils. Cell

Approximate Intravascular Life Span

Major Function

Erythrocyte (red cell)

120 days

Oxygen transport

Neutrophil

7-10 hours

Monocyte

Several days

Eosinophil

A little shorter than neutrophils

Defence against parasitic infection

Lymphocyte

Very variable

Humoral and cellular immunity

Platelet

10 days

Haemostasis

Defence against infection by phagocytosis and killing of microorganisms Defence against infection by phagocytosis and killing of microorganisms

2. Explain the haematopoietic origins of red blood cells, platelets and neutrophils, and list the main physiological factors that influence rate of red cell production. Blood cells of all types originate in the bone marrow and are all ultimately derived from pluripotent haemopoietic stem cells Pluripotent haemopoietic stem cells give rise to lymphoid stem cells (T cells, B cells, NK cells) and multipotent haemopoietic stem cells (granulocyte, monocytes, erythrocytes, megakaryocytes/platelets)

Red cells Red blood cells are produced under the influence of erythropoietin which is synthesised in the kidney (90% - juxtatubular interstitial cells), in response to hypoxia (10% hepatocyte and interstitial cells in liver)

Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Haematology Multipotent haemopoietic stem cell → proerythroblast → erythroblast (early/intermediate/late) → erythrocyte/RBC The process of production is called erythropoiesis The erythrocyte lasts about 120 days in the circulation - ultimately destroyed by the phagocytes of the spleen Principle functions are transport of oxygen and carbon dioxide Oxygen delivery is facilitated by the sigmoid oxygen dissociation curve and the fact that reduced pH lowers the oxygen affinity of haemoglobin. Haemoglobin also acts as a buffer. White Blood Cells (granulocytes and monocytes) Bone marrow production of granulocytes and monocytes is under the influence of multiple cytokines e.g. interleukins, granulocyte colony stimulating factors (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) The pluripotent haemopoietic stem cell → myeloblast/multipotent haemopoietic stem cell → granulocytes and monocytes The neutrophils granulocyte lasts 7-10hrs in the circulation before migrating to tissues Main function is defence against infection through phagocytosis Another product of the myeloblast are eosinophils granulocytes which spend less time in the circulation than a neutrophils and whose main function is defence against parasitic infection Basophil granulocytes have a role in allergic responses The multipotent stem cell also gives rise to monocyte precursors and so monocytes. Monocytes spend several days in the circulation Monocytes migrate to the tissues where they transform into macrophages and other phagocytic scavenging cells. Macrophages also store and release iron White Blood Cells (Lymphocytes) Pluripotent haemopoietic stem cell → lymphoid stem cell → T-cells, B-cells, NK-cells Lymphocytes recirculate through the lymph, lymph nodes, blood stream tissues The intra-vascular life span is very variable Platelets The production of bone marrow is under the influence of thrombopoietin Multipotent haemopoietic stem cell → megakaryocyte → platelets Platelets survive around 10 days in the circulation Have a role in primary haemostasis Platelets contribute phospholipid which promotes blood coagulation

3. State the approximate normal ranges for a standard blood count, and know that the normal ranges in children and people with African ancestry differ from those of adult northern Europeans. Reference ranges are descriptions of data derived from a sample of a reference population Commonly given as 95% ranges so that 2.5% is excluded at each end of the range If the data has a Gaussian distribution then the mean, plus or minus 2 standard deviations gives a 95% range A normal range is the reference range derived from healthy reference population. However a 95% range derived from an apparently healthy population includes data from patients with a high risk of developing significant disease It may be more relevant to interpret data in the light of whether a laboratory result is predictive of good future health rather than whether it falls within the 95% limits for apparently healthy people Normal is dictated by: o Age o Gender o Ethnic origin o Physiological status o Altitude o Smoking, drinking A value within normal range may be abnormal for that individual so it is always relevant to consider past results A value outside the normal range may be normal for that individual. By definition 5% of healthy subjects fall outside the normal range Reference ranges for healthy and sick normally overlap Some haematological variables are dependant on the precise instrument or methodology used. WBC – White Cell Count, the number of white cells in a given volume of blood (x 10 9 / L) RBC – Red Cell Count, the number of red cells in a given volume of blood (x 10 12 / L) Hb – Hb concentration (g/L or g/dL) PCV – Packed Cell Volume, the proportion of a column of centrifuged blood occupied by red cells (L/L) Hct – Haemocrit, equivalent to PCV (L/L) MCV – Mean Cell Volume, i.e. the average size of red cells (fl) MCV = PVCx1000 / RBC MCH – Mean Cell Haemoglobin, the average amount of Hb in a red cell (pg) how much Hb is related to size of cell

Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Haematology MCHC – Mean Cell Haemoglobin Concentration – the average concentration of Hb in a red cell (g/l or g/dl) – how concentrated the Hb is in each cell Platelet Count – the number of platelets in a given volume of blood (x10 9/L)

95% RANGES FOR CAUCASIAN ADULTS WBC RBC Hb PCV (Hct) MCV MCH MCHC Platelet count Neutrophils Lymphocytes Monocytes Eosinophils Basophils Reticulocytes

Males 3.6-9.2 x 109/l 4.25-5.77 x 1012/l 13.5-16.9 g/dl 0.41-0.51

84-99 fl 27.5-32.7 pg 30.9-34.8 g/dl

143-332 x 109/l 1.7-6.1 x 109/l

1.0-3.5 x 109/l 0.2-0.6 x 109/l 0.03-0.46 x 109/l 0.02-0.09 x 109/l 20-130 x 109/l

Females 3.5-10.8 x 109/l 3.82-4.98 x 1012/l 11.5-14.8 g/dl 0.36-0.46

169-358 x 109/l 1.7-7.5 x 109/l

4. Recognise the terms commonly used in describing abnormalities in blood counts and films and explain what they mean Anisocytosis – red cells show more variation in size than is normal Poikilocytosis – red cells show more variation in shape than is normal Microcyte – a red cell that is smaller than usual Microcytic anaemia – anaemia with small red cells Microcytosis – red cells are smaller than normal Macrocyte – a red cell that is larger than normal. May be round, oval or polychromatic Macrocytic anaemia – anaemia with large red cells Macrocytosis – red cells are larger than normal Normochromic – red cells with a normal sized central pallor Normocyte – a red cell of normal size Hypochromic – red cells that display hypochomia Hypochromia – red cells which have a larger central area of pallor caused by lower Hb content and concentration, and a flatter cell In normal cells about a third of the diameter is pale. Often goes together with microcytosis Hyperchromia – cells lack central pallor due to being thicker than normal or an abnormal shape Polychromasia – describes an increased blue tinge to the cytoplasm of a red cell, shows the cell is young Elliptocyte – red cells that are elliptical in shape. Occur in hereditary elliptocytosis and iron deficiency. An axample of poikilocytosis Spherocytes – cells that are approximately spherical in shape. Hyperchromic, lacking central pallor with a round, regular outline. Are caused by a loss of cell membrane without a loss of an equivalent amount of cytoplasm so the cell is forced into a spherical shape. An example of a poikilocyte Target cell – cells with an accumulation of Hb in the central pallor. They occur in obstructive jaundice, liver disease, haemoglobinopathies and hyposplenism. An example of a poikilocyte Sickle cell – red cells which are sickle or crescent shaped. Result from the polymerisation of Hb S. An example of poikilocytosis Fragment – also known as schistocytes. Are small fragments of red cells indicating fragmentation of red cells Rouleaux – stacks of red cells resembling coins resulting from alterations in plasma proteins Aggutination – irregular clumps of red cells usually resulting from antibodies on cell surface Howell-Jolly body – a nuclear remnant in a red blood cell, commonly caused by lack of splenic function Leucocytosis – too many white cells Leucopenia – too few white cells Neutrophilia – too many neutrophils Neutropenia – too few neutrophils Lymphocytosis – too may lymphocytes Atypical lymphocyte – an abnormal lymphocyte, often present in infectious mononucleosis. Atypical mononuclear cell is another term used Eosinophilia – too many eosinophils Monocytosis – too many monocytes Thrombocytosis – too many thrombocytes

Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Haematology Thrombocytopenia – too few thrombocytes Toxic granulation – heavy granulation of neutrophils, results from infection, inflammation, necrosis and pregnancy Left shift – means that there is an increase in non-segmented neutrophils or that there are neutrophils pre-cursors in the blood Hypersegmented neutrophil – an increase in the average number of neutrophil lobes or segments usually resulting from a lack of Vit. B12 or folic acid Reticulocytosis – a way to detect young red cells is to do a reticulocyte stain which causes methylene blue to precipitate as a network or reticulum in living cells. Similar to polychromasia.

Mechanisms of Anaemia and Polycythaemia 1) Explain the term anaemia Anaemia is a reduction in the concentration of Hb in the circulating blood below what is normal for a healthy individual of the same age and gender as the individual. Anaemia is usually associated with a reduction in the red blood cell count (RBC) and haemocrit (Hct) or packed cell volume (PCV)

2) Describe the mechanisms underlying the development of anaemia Causes and mechanisms of anaemia are distinct: The mechanism of anaemia may be the reduced production of haemoglobin in the bone marrow The cause of anaemia could be either a condition relating to a reduction in the production of haem or globin Mechanisms of anaemia: o Reduced production of red cells by the bone marrow o Loss of blood from the body o Reduced survival of red cells in the circulation (haemolysis) o Increased pooling of red cells in an enlarged spleen

3) Explain how anaemias are classified on the basis of red cell size Anaemias can be classified not only by mechanism but also by the size of the red cells present. They are classified as either: o Microcytic (small and usually hypochromic) o Macrocytic (large and usually normochromic) o Normocytic (normal and usually normochromic) Classification by size can help suggest an underlying cause of the anaemia

4) List the common causes of microcytic, normocytic and macrocytic anaemia Microcytic Red cells are small - referred to as microcytes Size is measured visually on a slide or by looking for a reduced Mean Cell Volume (MCV) Microcytic cells are usually hypochromic and so appear pale and have a reduced MCHC (Mean Cell Hb Concentration) Microcytosis results from reduced synthesis of haemoglobin, either by reduced haem, or reduced globin synthesis The common causes are: o Iron deficiency anaemia (haem) o Anaemia of chronic diseases (haem) o Thalassemia – an inherited defect leading to reduced globin synthesis Macrocytic Red cells are larger than normal - referred to as macrocytes Cells have an increased MCV Usually results from abnormalities in haemopoiesis so that red cell precursors continue to synthesise Hb and other cellular proteins but fail to divide properly One cause of macrocytic anaemia is megaloblastic erythropoiesis. This refers specifically to the delay in the maturation of the nucleus while the cytoplasm continues to grow and mature A megaloblast is an abnormal bone marrow cell which is larger than normal and shows nucleo-cytoplasmic dissociation An alternative mechanism of macrocytic anaemia is premature release of cells from the bone marrow Premature cells (reticulocytes) are approx 20% bigger – more in circulation, average cell size will increase (MCV) Important causes of macrocytic anaemia include: o Megaloblastic anaemia resulting from Vit. B12 or folic acid deficiency. o Use of drugs interfering with DNA synthesis o Liver disease o Excess alcohol intake o Recent major blood loss with sufficient iron stores leading to release of reticulocytes o Haemolytic anaemia leading to increased reticulocyte production

Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Haematology Normocytic Cells normal size and usually normochromic (normal staining) Important causes include; o Recent Blood loss – peptic ulcer, trauma, oesophageal varices o Failure in production of red cells – renal failure, bone marrow failure or suppression, bone marrow infiltration, early stages of IDA, AoCD o Hypersplenism – pooling of red cells in the spleen

5) List the causes of haemolytic anaemia and describe how it is recognised Haemolytic anaemia is anaemia due to the shortened survival of red cells in the circulation Can be acquired or inherited – intrinsic abnormality of red cells or extrinsic factors affecting red cells Extrinsic factors can interact with intrinsic abnormalities Intravascular haemolysis results from very acute damage and destroys cells in the circulation Extravascular haemolysis results when defective cells are removed by the splenic macrophages Often haemolysis involves both parts

INHERITED

ACQUIRED

Abnormal red cell membrane, e.g. hereditary spherocytosis

Damage to red cell membrane, e.g AIHA or snake bite Damage to whole red cell, e.g, MAHA

Abnormal Hb, e.g. sickle cell anaemia

Oxidant exposure, damage to red cell membrane and Hb, e.g. dapsone or primaquine

Defect in glycolytic pathway, e.g. pyruvate kinase deficiency

Defect in enzymes of pentose shunt, e.g. G6PD deficiency

Precipitation of episodic haemolysis in individuals with enzyme deficiency

When to suspect haemolytic anaemia: o Otherwise unexplained anaemia which is normochromic and usually normocytic or macrocytic o Evidence or morphologically abnormal red cells – e.g. spherocytes, elliptocytes, fragments o Evidence of increased red cell breakdown – e.g. increased unconjugated serum bilirubin, and lactate dehydrogenase o Evidence of increased bone marrow activity – e.g. polychromasia → increased reticulocyte count Inherited:

Site of defect

Examples

Membrane

Hereditary spherocytosis

Haemoglobin

Sickle cell anaemia

Glycolytic pathway

Pyruvate kinase deficiency

Pentose shunt Acquired:

Glucose-6 phosphate dehydrogenase deficiency

Site of defect and nature of damage

Examples

Membrane – immune

Autoimmune haemolytic anaemia

Whole red cell – mechanical

Microangiopathic haemolytic anaemia

Whole red cell – oxidant

Drugs and chemicals

Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Haematology Whole red cell – microbiological

Malaria

Hereditary Spherocytosis - after entering the circulation the cells lose membrane in the spleen and thus become spherocytic. The cells become less flexible and are removed by the spleen – extravasulvar haemolysis. The bone marrow responds by increased production leading to increased numbers of reticulocytes and polychromasia. The haemolysis leads to increased bilirubin excretion, jaundice and gallstones. The only effective treatment is a splenectomy, a good diet, or folic acid treatment is required to prevent folic acid deficiency Glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency) is caused by a lack of G6PD which is an essential enzyme in the pentose phosphate shunt which is essential to stop cellular damage by exogenous oxidants. The gene for G6PD is on the X chromosome so sufferers are normally hemizygous males (occasionally homozygous females). Oxidants that can lead to haemolysis in sufferers; sources include the broad bean, naphthalene and certain drugs. The deficiency leads to intermittent severe intravascular haemolysis as a result of infection or exposure to an oxidant such as those named above. Haemoglobin is denatured and forms round inclusions known as Heinz bodies, which can be detected by a specific test. Heinz bodies are removed by the spleen, leaving a defect in the cell – irregularly contracted cells → intravascular heamolysis. Autoimmune haemolytic anaemia results from production of autoantibodies directed at red cell antigens. The immunoglobulin bound to the red cell membrane is recognized by splenic macrophages, which remove parts of the red cell membrane, leading to spherocytosis – further removed by the spleen

6) Explain the possible mechanisms underlying polycythaemia Polycythaemia is the opposite of anaemia and literally means “too many” cells The Hb, RBC and PCV/Hct are all increased compared to the normal Pseudopolycythaemia is caused by a decrease in plasma volume True polycythaemia is caused by an increase in the numbers of circulating red blood cells Classification of polycyhtaemia: o Physiological – e.g. in a new born baby o Appropriate erythropoietin secretion – e.g. residence at high altitude, or in hypoxic patients o Inappropriate erythropoietin secretion – e.g. abuse by athletes, erythropoietin secrested by renal tumours or cysts o Not mediated by erythropoietin but due to intrinsic bone marrow disease – polycythaemia vera It can lead to thick blood or hyperviscosity which can lead to vascular obstruction Blood can be removed to thin the blood, or drugs given to reduce bone marrow production

Haemostasis and Abnormalities of Haemostasis 1) Describe the normal haemostatic mechanism including the interactions of vessel wall, platelets and clotting factors Vessel wall contraction Occurs as a local contractile response to injury This in itself is sometimes enough to temporarily restrict blood flow in smaller vessels. Most important in small vessels, the mechanism is not really understood Formation of an unstable platelet plug Platelet adhesion and aggregation Immediately following injury platelets adhere to the subendothelial structures Mediators of binding include extravascular collagen, von Willibrand Factor, and membrane glycoproteins Activation of the platelets - synthesis of ADP and thromboxane An important metabolic pathway in platelets converts membrane phospholipids to thromboxane A2, which is a potent inducer of platelet aggregation (Membrane phospholipids → Arachidonic Acid → Endoperoxides → TXA 2) Drugs such as aspirin target the metabolism pathway enzyme cyclo-oxygenase

Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Haematology Platelet adhesion Von Willebrand factor

platelet Endothelial cells

platelet

GlpIb GlpIa collagen

Release of ADP & thromboxane

Coagulation activation

thrombin

Platelet aggregation

GlpIIb/IIIa platelet

platelet

Fibrinogen + Ca2+

Stabilisation Stabilisation of the plug with fibrin Tissue factor activation of the blood coagulation system and the formation of a fibrin clot The main initiator is tissue factor which is exposed by vessel damage and forms an activation complex with Factor VIIa Tissue factor also activates the intrinsic pathway via Factor IX Factors VII, IX, X and prothrombin bind to phospholipids in order to activate their substrate factor. Phospholipid binding requires Vitamin K dependant post translational modification of certain amino acids and is mediated by Ca2+ Factors VIII and V, once activated accelerate these membrane dependant reactions on the platelet surface, enabling clotting to be localised to the site of injury

XII

XIIa

XIa

Not important for normal haemostasis

Blood coagulation

IXa

IX INTRINSIC PATHWAY

Xa Prothrombin

EXTRINSIC PATHWAY

VIIa Ca2+

VIIIa Pl Ca 2+ X

Tissue factor (vessel damage)

VIIa Ca2+

Va Pl Ca2+

X COMMON PATHWAY

thrombin (IIa)

Fibrinogen

Fibrin thrombin XIIIa XIII Crosslinked fibrin

Role of Platelet in Accelerating Blood Coagulation

thrombin Xa IXa Ca 2+

Ca 2+

Xa II Va

IXa X VIIIa

Va, VIIIa, platelets accelerate thrombin generation 10, 000 fold

Platelet membrane phospholipid surface

Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Haematology Dissolution of the clot and vessel repair Formation of the clot facilitates the activity of the main plasminogen activator, tissue plasminogen activator (tPA) The clot is lysed by the fibrinolytic enzyme system (fibrinolysis), and blood vessel repair is initiated Antithrombin and the Protein C anticoagulant pathway using Protein C and Protein S, act as coagulation inhibitory pathways and prevent blood clotting completely when vessels are damaged

Fibrinolysis

Plasminogen

tissue plasminogen activator, tPA

Plasmin

Fibrin clot

tPA and a bacterial activator, streptokinase, are used in therapeutical thrombolysis for Myocardial Infarction (Clot busters)

Fibrin degradation products, FDP -elevated in DIC

Platelets Platelets have their origin in the bone marrow Haemopoietic stem cells → megakaryocyte precursors which then undergo nuclear replication, maturation and granulation. They then migrate to the marrow sinusoids where they fragment and form platelets Each megakaryocyte produces approx 4000 platelets Lifespan of a platelet = 10 days - 1/3 of platelets are stored in the spleen

2) Describe and distinguish the clinical features of bleeding due to thrombocytopenia and coagulation disorders respectively Disorders of primary haemostasis Disorders of platelets o Number = thrombocytopenia o Function o Common causes such as drugs o More rare causes such as Glanzmann’s disorder Disorders of von Willibrand Factor o Quantity = Type I VWD o Function = Type II VWD Disorders of the vessel wall o Rare collagen defects such as Ehlers Danlos o Scurvy, old age, steroid therapy Thrombocytopenia – a disorder of number Failure of platelet production by megakaryocytes: o Generalised marrow failure – aplastic anaemia, viruses, drugs, leukaemia, B12/folate deficiency o Specific failure of megakaryocytes – myelodysplasia, congenital syndromes Shortened half-life of platelets: o Immune thrombocytopenia purpura  Auto-Immune Thrombocytopenic Purpura (auto-ITP) is a very common cause of thrombocytopenia. Antiplatelet anti-bodies attached to, and sensitise platelets which are then destroyed by macrophages o Immune stimulated by drugs o Coagulation consumption – disseminated intravascular coagulation Increased pooling of platelets in the spleen - hypersplenism Disorders of platelet function: Acquired: common; caused by drugs such as aspirin and other NSAID (COX inhibitors) Congenital: very rare; caused by surface glycoprotein deficiency or storage pool disease Bleeding in disorders of primary haemostasis (e.g. thrombocytopenia, VWD, reduced platelet function)

Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Haematology Easy bruising, superficial bleeding into skin, mucosal membranes Prolonged nosebleeds of more than 20mins Prolonged gum bleeding Petechiae (small red spots on skin) in thrombocytopenia Menorrhagia Prolonged bleeding after trauma and surgery Disorders of coagulation The role of the coagulation cascade is to produce a burst of thrombin which will convert fibrinogen to fibrin Deficiency in any of the coagulation factors results in failure of thrombin generation and hence fibrin generation In small vessels, vessel constriction is sufficient to stop bleeding, in larger vessels the formation of a stabilised fibrin clot is required Haemophilia – a disorder of secondary haemostasis o X-linked recessive o Haemophilia A & B o Factor VIII deficiency o Factor IX deficiency o All are clinically indistinguishable All other bleeding disorders are autosomal recessive – 50% of function is normally sufficient so person must by homozygous to be affected Bleeding in coagulation disorders Superficial cuts do not bleed as no disorder of platelet function Bruising is common, nosebleeds are rare Spontaneous bleeding is deep into muscles and joints - haemarthrosis Bleeding after trauma may be delayed and is prolonged Bleeding may restart after stopping

3) Describe the use of laboratory tests to assess haemostasis Tests to monitor platelets and their function Platelet count: o Used to monitor thrombocytopenia o Below 10 x109/L you get severe spontaneous bleeding – this occurs during treatment of leukaemia o Below 40 x109/L spontaneous bleeding is common = auto-ITP o Below 100 x109/L there is no spontaneous bleeding, but bleeding after trauma o Normal range is 150-400 x109/L Bleeding Time: o Cuff with 40mmHg pressure o Standardised incision o Bleeding time should normally be 3-8 min o Used to check platelet-wall interaction when platelet count is normal Platelet Aggregation: o Measures the functional defect of platelets o Used in vWD and inherited platelet disorders Tests of coagulation function APTT – Activated Partial Thromboplastin Time o Initiates coagulation through Factor XII and detects abnormalities in Intrinsic and Common pathways Pt – Prothrombin Time o Initiates coagulation through tissue factor and detects abnormalities in Extrinsic and common pathways APTT and PT are used together for screening for causes of bleeding disorders APTT is used for monitoring heparin therapy in thrombosis PT is used for monitoring warfarin treatment in thrombosis

4) Describe the principles of management of disorders of haemostasis Vessel Wall If acquired then treat underlying condition such as hypersensitivity reaction that requires the steroids that then cause bleeding disorder If inherited then use local treatment such as nasal packaging for nosebleeds Very difficult to treat Platelets If failure is in production or function then replace with platelet concentrates and stop taking drugs (the action of aspirin takes

Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Haematology several days to wear off) If failure is due to consumption then take steroids for immuno suppression of ITP(immune thrombocytopenic purpura). If due to pooling in spleen then carry out a splenectomy Coagulation and VWD If inherited then replace the missing coagulation factor such as Factor VIII If acquired then take FFP (Fresh Frozen Plasma) for multiple factor deficiency In factor replacement therapy all factors are available except for Factor V DDAVP = Desmopressin o Vasopressin derivative which leads to a 2-5 increase in vWF-Factor VIII by replacing endogenous stores o Only useful in mild disorders Tranexamic Acid o A lysine derivative which binds to plasminogen and blocks binding to fibrin and so reduces clot lysis. It is useful as an adjuvant therapy and can cross the placenta Heparin o Used as an immediate anticoagulation therapy in venous thrombosis and pulmonary embolism o Acts by accelerating the action of the plasma inhibitor antithrombin. Warfarin o Used for long term anticoagulation following thrombosis o Acts by inhibiting production of thrombin within platelets by inhibiting Vit. K and Ca2+ dependant pathways

Blood Transfusion 1) Describe the major significant blood groups and their importance clinically The ABO system is important because people have naturally occurring antibodies that are IgM, reactive at 37ºC and capable of activating complement. They are, therefore, able to cause haemolysis if incompatible blood is transfused → fatal Frequency in population (UK)

Blood group

Genes

Antigens

Antibodies

46%

nil

anti-A and anti-B

43%

AO/AA

anti-B

BO/BB

anti-A

A and B

nil

A and B antigens are created on blood cells by adding one or other sugar residue onto a common glycoprotein and fructose stem on the red cell membrane Group O has neither A or B antigens and just have the glycoprotein and fructose stem A group gene codes for the addition of N-acetyl galactosamine to the common stem B group genes code for the addition of galactose to the common stem There are also other important antigens on the cell surface including the Rh antigens (C, D, E and K), Kell, Duffy All can form Ab’s (IgG) so in subsequent transfusions negative blood must be used to prevent a delayed haemolytic reaction Genes for RhD groups (most important) – D codes for D antigen and is dominant, d codes for no antigen and is recessive HDN – Haemolytic disease of the newborn. If an RhD negative mother has been sensitised and then has an RhD positive fetus the maternal IgG antibodies can cross the placenta and cause fetal haemolysis which can cause death Frequency in population (UK)

Blood group

Antigens

Antibodies

85%

RhD positive

15%

RhD negative

nil

after exposure to RhD pos blood fetus can make anti-D

O neg is the universal donor blood, but only 6-7% of the population are O neg Blood grouping – patients red cells incubated with known antibodies, observing for agglunation Antibody Screen – exclude any clinically significant immune antibodies. Recipient serum 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. If the screen is negative, any donor blood which is ABO (and D) compatible can be given. If positive, the antibody must be identified with the use of a large panel of red cells; donor units that lack the corresponding blood group antigen are then chosen for cross matching with the recipient's serum prior to transfusion. Cross match – a compatibility test is done between donor red cells and recipient serum

Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Haematology 2) Describe the screening of blood donors undertaken and reasons why Donors are excluded if they have any disease that may make giving blood hazardous, e.g. cardiovascular or neurological disease Donors are tested for certain infections, and question/excluded due to behaviours that may put the recipient at risk Common causes for donor exclusion (tests for antigens not 100% accurate): o Men and women who are infected with HIV o Men who have had sex with any other man since 1979 o Men and women who have used IV drugs o Men and women who have had sex at any time since 1977, with men or women who have lived in African countries, except those bordering the Med o Men and women who have had sex with anyone in the above groups o Men and women whose sexual partners are haemophiliacs o Men and women who are prostitutes o Men and women who have had sex with a prostitute All blood is tested for: (some tests are for the antigen (Ag) and some for the antibody (Ab)) o Hep B (Ag) o Hep C (Ab) o HIV (Ab) o HTLV (human T-lymphotrophic virus) o Syphilis (Ab) o CMV (cytomegalovirus) Some viruses could be transmitted that cannot or are not tested for such as malaria, so questioning excludes at risk donors

3) Describe the various blood components used and the potential side effects of blood transfusion Blood is split to give more efficient use and longer storage times The blood is split via centrifuge which puts red cells at the bottom, platelets in the middle, and plasma at the top. It is then cut into satellite bags To avoid the theoretical risk of CJD through transfusion in the UK: o Plasma from UK donors is no longer used for fractionation o All blood products are LEUCODEPLETED to remove white blood cells 1 UNIT = WHOLE BLOOD OR BLOOD PRODUCTS DERIVED FROM ONE SINGLE BLOOD DONATION

W h o le B lo o d

R BC S A G -M

R e d C e lls (R B C )

P la t e le t s

P la s m a re d u c e d R B C

F ro z e n R BC

P la s m a

FFP

C r y o p r e c ip it a t e + C ry o s u p e rn a ta n t

P la s m a f o r F r a c t io n a t io n A lb u m in F V III F IX I m m u n o g lo b u lin s A n t it h r o m b in I I I e tc .

Whole Blood Less than 1% of blood used in the UK Is deficient in labile clotting factors and functional granulocytes and platelets Red Cells 1 unit from 1 donor of packed cells (plasma removed) Shelf life of 5 weeks at 4°c in a fridge Given through a blood giving set to filter out clumps and debris There is the National Frozen Bank for rare groups/antibodies but use is rare and recovery is poor on thawing Platelets Available in two forms: o Pooled platelets – platelets from several (4-5) donations pooled to constitute a single adult dose o From a single donor by cell separator machine, equivalent to 4-5 single donations of platelets Stored at room temp (22°c) Shelf life of only 5 days due to risk of bacterial infection Need to know blood group. It does not need to be fully cross matched but platelets do have low levels of ABO and RhD antigens One pool is generally enough Indications for use: o Preventative – prohylaxis due to thrombocytopenia or defective platelet function.

Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Haematology o Therapeutic – for treatment of bleeding due to thrombocytopenia or dysfunction In autoimmune thrombocytopenia platelets transfusions are rarely indicated due to rapid destruction of platelets White Cells Very rarely used except in severe infections that occur in neutropenic patients not responding to antibiotics/antifungals FFP (Fresh Frozen Plasma) 1 unit from 1 donor (300ml) Shelf life of 1 year at -30°c, frozen within 6hrs to preserve clotting factors Must thaw approx 20-30mins before use and then must be used within 1hr or clotting factors will degrade Standard dose is 12-15ml/kg, so one unit normally enough ABO and RhD compatible FFD must be used as there can be minimal red cell contamination and presence of ABO antibodies/RhD sensitisation AB is universal donor for plasma as no A or B antibodies Indications for use: o If bleeding with abnormal coagulation tests (APTT, PT) o Reversal of Warfarin for immediate surgery o Is not used to replace fluid/volume Cryoprecipitate Separated from other plasma constituents by freezing fresh plasma and then allowing it to thaw at 4-8°c overnight Approx 3% of the FFP forms a precipitate and fails to redissolve = cryoprecipitate Contains factor VIII and fibrinogen Stored as frozen in small volumes (approx 15ml) When thawed quickly for use it redissolves in plasma Shelf life of approx I year Standard dose from 10 donors Indications for use: o Massive bleeding with low fibrinogen o Fibrinogen deficiency Fractionated Products Made from imported plasma from US (vCJD risk) Albumin – Human Albumin Solution (HAS) = 4.5%. A safe product that is pasteurised and never been implicated in transmission of infection. Used in burns, major surgery and plasma exchanges. Few uses, overused. 20% in salt poor for treating certain liver conditions Factor VIII Concentrate – large pools of plasma (2,000-5,000 donations). Heat treated to eliminate viral transmission. Used in the treatment of haemophilia A and in vWD and a prophylaxis and to control massive bleeding. Recombinant Factor VIII is now given to all young haemophiliacs in the UK Factor IX Concentrate – Used in treatment of Christmas disease and Haemophila B. Recombinant IX also available Normal Human Immunoglobulin – prepared from pooled human plasma and contains a mixture of Ig present in healthy adult population. Given as IM or IV in treatment of immunodeficiency states and prevention of certain disease such as measles and Hep A Specific Immunoglobulin – fractionated from plasma from specific donors who have a high titre of specific immunoglobulin e.g. anti-D Ig, Hep B, Rabies, Tetanus etc. Used to treat specific diseases Adverse Effects Immediate complications of transfusion (within 1-2hrs). ABO incompatibility and bacterial infection are the two commonest causes of death: Immunological Haemolytic transfusion reaction (ABO incompatibility reaction) Febrile, non-febrile reaction Utricarial rash Analphylactic reaction Transfusion related acute lung injury (TRALI) Delayed complications of blood transfusion:

Non - Immunological Bacterial contamination – endotoxic shock Congestive cardiac failure (overload) Hypothermia, Hyperkalaemia, Hypocalcaemia in large volume transfusions Air embolism if air in tubing (rare)

Immunological Delayed haemolytic transfusion reactions (other blood group antibodies) Post-transfusion pupura Graft-versus-host disease Immunomodulatory effects (increased patient infections)

Non-Immunological Viruses – Hep B and C, HIV 1 and 2, HTLV, Parovirus, CMV etc Other – Malaria, Syphilis etc Iron overload

Autologus Blood – sometimes a patient can have their own blood instead of a donor. Two types:

Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Haematology Pre-operative autologus blood – patients donate a few units of their own blood 5 weeks prior to surgery Cell salvage – During large, bloody operations the patient’s blood can be salvaged washed and centrifuged before being given back provided area of operation is not contaminated

Sickle Cell Disease 1. Describe the inheritance of clinical and haematological features of sickle cell anaemia (SS) • • • • • •

•

• • • •

Up to 10% Caribbeans and 25% Africans (sub-Saharan) carry sickle gene Distribution matches that of endemic Plasmodium falciparum malaria Around 200,000 affected births annually worldwide Point mutation at codon 6 of the gene for β globin Glutamic acid is replaced by valine Glutamic acid • Polar • Soluble Valine • Non polar • Insoluble Deoxyhaemoglobin is insoluble These molecules can then polymerise within the red cell, which distorts and undergoes a characteristic shape change: the sickled cell These cells have a marked decrease in deformability. In addition, the formation of intracellular polymers is associated with red cell membrane changes, which make the red cells particularly “sticky” to vascular endothelium Rigid, adherent, dehydrated

Sickle cell disease

Condition

β genes

Haemoglobins present (in addition to A2 and F)

Sickle cell anaemia

βSβS

Sickle cell/haemoglobin C disease

βSβC

S and C

S A

Sickle cell trait

ββ

A and S

Normal

ΒAβA

Only adult Hb is affected because HbF does not have any beta chains – problem starts at 4-6 months or older As sickled red cells become trapped in the small blood vessels, circulation is impaired and there is damage to multiple organs In children, infarcts of the small bones of the hands of feet may occur and lead to a painful dactylitis called the “hand-foot“ syndrome and, as a later result, shortening of the digits In adults, generalised pains are more typical and result from oxygen deprivation of tissues and avascular necrosis of the bone marrow Bones dactylitis / osteomyelitis/ avascular necrosis of the hip Kidneys haematuria and failure to concentrate urine, papillary necrosis Brain stroke Lungs “chest crisis” Spleen splenic sequestration/ hyposplenism Skin skin ulcers Painful crises triggered by o Infection o Exertion and psychological stress o Dehydration o Hypoxia Terminology Infarct - death of tissue due to loss of blood supply Dactylitis - inflammation of a digit (in this case resulting from infarction of bone) Avascular necrosis - death of tissue as a result of loss of its blood supply Osteomyelitis - infection of bone (dead tissue is susceptible to bacterial infection) Splenic sequestration - pooling of large numbers of red cells in the spleen (see below)

Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Haematology Hyposplenism - reduced function of the spleen (in this case, as a result of recurrent interruption of the blood supply leading to death of splenic tissue) ‘Chest crisis’ - hypoxia resulting from death of lung tissue Sickled cells are fragile and have a shortened life span (haemolysis), which results in anaemia The affinity of HbS is lower than that of HbA so it gives up oxygen more readily to tissues and anaemia is often well tolerated In an attempt to compensate for the shortened red cell life span there is an increased turnover of red cells and the body’s supply of folic acid can become low Particularly venerable to parvovirus B19 infection - virus infects red blood cell precursors and stops red cell production for up to a week. In the setting of a short red cell life span, if red blood cell production stops for even short period of time the Hb level can fall dramatically; this is called an aplastic crisis. Children are also at risk of another sort of crisis called a splenic sequestration crisis -abdominal pain, pallor and shock together with a large spleen and low haemoglobin are indicative. The reticulocyte count is raised in a sequestration crisis but in an aplastic crisis it is much lower than normal. Diagnosis The blood count shows a low Hb e.g. 6-9 g/dl and raised reticulocyte count Blood film shows sickled cells. Also it may show signs of hyposplenism, namely the presence of Howell-Jolly bodies (which are nuclear remnants usually removed in the spleen) and target cells. Simple screening tests. o These tests depend on the decreased solubility of haemoglobin S when the oxygen tension is low o One such test is a sickle solubility test in which a reducing agent is added to diluted blood and leads to formation of many sickle cells (in sickle cell trait as well as in sickle cell anaemia) o This makes the blood turbid. A positive result must be confirmed by Hb electrophoresis. Definitive diagnosis requires haemoglobin electrophoresis (or an equivalent test) as well as a sickle solubility test. Electrophoresis separates proteins according to their charge; this varies according to the pH at which electrophoresis is carried out. So, at alkaline pH, HbS separates readily from Hb A and F. However, there are some non-sickling haemoglobins (called HbD and HbG) that run with HbS – this is why a sickle solubility test is also needed. o In sickle cell anaemia no HbA is detected and there is a variable (5-15%) amount of HbF and a small amount of HbA 2 o Patients with sickle trait have Hbs A and S (plus small amounts of HbA 2 and HbF)

2. Outline principles of management Painful crisis o Factors known to precipitate a crisis should be avoided. Fast, adequate pain relief with strong analgesics should be given and precipitating factors should be reduced by rehydration, warmth and additional oxygen as necessary. Infection should be excluded by chest X-ray and appropriate cultures, e.g. of urine and blood. If infection is present, antibiotics are needed. Folic acid 5mg /day Vaccination to protect against pneumococcal infection Prophylactic penicillin to prevent some of the infections caused by hyposplenism Blood transfusion o Top up blood transfusion e.g. if aplastic or sequestration crises o Exchange blood transfusion if life threatening/severe disease such as a stroke, or chest crisis. An exchange transfusion aims to reduce the HbS to less than 20% o Top-up blood transfusion is NOT a treatment for painful crisis; it will increase blood viscosity and may make the painful crisis worse Stem cell transplantation - consider in children with severe disease. Currently survival is 90-95%.

3. Explain the inheritance, clinical significance and diagnosis of sickle cell trait Carrier state for sickle cell disease Does not affect life expectancy, and is often clinically silent Needs to be identified because certain situations can provoke sickling e.g. anaesthesia, high altitude, air travel in unpressurised planes All patients from ethnic groups in whom βS occurs should be screened prior to surgery Sickle trait must also be identified in pregnant women so that their partners can be tested and appropriate counselling given and action taken if necessary.