The Mature Red Cell Dunedin Basic Medical Sciences Course CELLULAR HAEMATOLOGY ► Flexible biconcave disk ~7-8 um diam ► Cytoplasm ▪ Viscous haemoglobin solution 320-350 g/L ▪ Hb packaged in red cells to minimise viscosity of blood ▪ Compare concentration with plasma ~70 g/L Presented by Prof Ian Morison FRCPA(Haem) Slides by Dr Jim Faed ▪ Energy handling ▪ Glycolytic (Embden-Meyerhoff) pathway ▪ Hexose monophosphate shunt ▪ No mitochondria, endoplasmic reticulum or ribosomes in mature red cells 2 1 Blood – the transport medium ► Normal Haematopoiesis blood volume ▪ Various formulae – simplest: mL/kg 95% ranges + 2sd Red cell volume ► Plasma volume ► Total blood volume ► ► men 25-35 40-50 65-85 women 20-30 40-50 60-80 Blood volume (mL/kg) with varying body build Gender Obese Thin Normal Muscular Male 60 65 70 75 Female 55 60 65 70 Neonate 100 3 Normal bone marrow 4 Haemopoietic cell lineages Mixed myeloid progenitor stem cell Pluripotent stem cell (Haemopoietic stem/progenitor cell) Lymphoid stem cell Erythroid progenitor (red cells) 5 Megakaryocyte progenitor Granulocyte (platelets) progenitor Monocyte progenitor Dendritic cell progenitors (antigenpresenting cells) B cell T cell NK cell progenitors 6 Hemopoiesis - regulation Haemopoiesis - regulation ► Control mechanisms - ▪ Cytokine signals ▪ induce growth and differentiation ▪ produced by peripheral tissues and cells of marrow ▪ Marrow stromal cells ▪ cell surface signals S Haemopoiesis is an integrated process ▪ Results in self renewal of pluripotent stem cells ▪ Differentiation of some pluripotent stem cells into specific cell lineages - irreversible 7 8 Regulatory signals in haemopoiesis Haemopoiesis Pluripotent stem cell (Haemopoietic stem cell) Mixed myeloid progenitor stem cell Erythroid Progenitor Regulation of cell pools by cytokines Lymphoid& stem cell marrow stromal cells Megakaryocyte Progenitor Granulocyte Progenitor Blood Red cells Blood Platelets Monocyte progenitor Blood Neutrophils Monocytes Basophils Eosinophils Dendritic cell progenitors Lymphoid tissues, skin, etc 1 2 3 B cell T cell NK cell Proliferation progenitors and differentiation of Thymus Bone committed cells Act on stromal cells ▪ IL-1, TNF, others Act on pluripotent stem cells ▪ ▪ Stem cell factor (SCF) FLT ligand ▪ IL-3, GM-CSF, G-CSF, IL-6, TPO Act on multipotential progenitor cells • Act on committed progenitor cells ▪ G-CSF - neutrophils ▪ M-CSF - monocytes ▪ IL-5 - eosinophils ▪ EPO - red cells ▪ TPO - platelets ▪ (plus IL-3 & GM-CSF) marrow Lymphoid tissues & marrow 9 Erythroblasts 10 Erythropoiesis - committed cells ► Proerythroblast 1-2d erythroblast 1-2 days ▪ rapid division, iron uptake ► Polychromatic erythroblast 3-4 days ▪ divides, Hb synthesised, then starts clearance of RNA, mitochondria, etc, and buds off nucleus to become a ... ► Reticulocyte 3-4 days ▪ immature red cell: completes clearance of RNA mitochondria, golgi, etc ▪ migrates into blood after 1-3d Marrow stages - 8-9 days ► Erythrocyte ~120 days ► Basophilic Polychromatic erythroblasts & neutrophil Key issues: Polychromatic erythroblasts 1. Proliferation surrounding a macrophage 2. Iron uptake 3. Haemoglobin production 4. and finally, removal of mitochondria, ER, ribosomes, golgi and loss of nucleus. 11 12 Expansion of cell numbers proerythroblasts 1-2 days ! ! 3-4 days ! ! basophilic erythroblasts ~4 days reticulocytes ! polychromatic erythroblasts { mature erythrocytes RNA and remnants of other cell organelles in young red cells are precipitated by new methylene blue dye, resulting in a blue reticulated pattern. Erythropoiesis haemopoietic stem cells 1-2 days Reticulocytes Manual (historical) retic count Normal: 1-2% ~ 20-90x109/L Current (large) haematology analysers are able to provide accurate reticulocyte counts and identify the proportion of young reticulocytes (increased in haemolysis) nucleus lost migrate into blood Mature erythrocytes - circulate ~120 days Red cells produced by an average adult: ~2.3 x 1011/day 13 Obtaining diagnostic bone marrow specimens (This film is from a case of haemolytic anaemia and has a raised reticulocyte count.) Automated retic count by fluorescence 14 Bone marrow biopsy ▪ Aspirates – for cytology ► Morphology of haemopoietic cells ▪ Recognise abnormal maturation ► Detection of malignancy (if >5% blast cells) ▪ Cytochemistry – films ▪ Immunological detection of cell surface markers to characterise acute leukaemias ▪ Trephine biopsy for histology ► Assess marrow stroma lymphoid and other malignancies in marrow ► Identify metastatic tumour deposits ► Detect Patient lying in recovery position 15 Red cell indices: NZ reference ranges Newborn – 22 d–3 m cord blood 7-10 y Female adult Male adult RBC 3.5-6.7 2.8-4.3 3.5-5.5 3.9-5.6 4.5-6.5 Hb (g/L) 137-201 95-130 101-139 115-165 130-180 MCV (fL) 99-117 72-90 76-96 MCH (pg) 30-37 26.33 80-98 27-32 17 Red Cell indices: international data Adult males NZ UK USA RBC (x1012/L) 4.5-6.5 4.5-6.5 4.3-5.9 Hb (g/L) 130-180 135-175 135-172 Ht 0.40-0.54 0.40-0.52 0.39-0.49 MCV (fL) 78-98 80-95 76-100 MCH (pg) 27-32 27-34 27-33 18 How to approach the red cell indices ▪ Carbohydrate (10%) - on external surface ▪ Lipid bilayer (40%) - fluid at 37oC ▪ Protein cytoskeleton (50%) Examples Evaluate the Red Cell Indices in this sequence - A B C D 4 RBC 4.4 5.0 4.8 3.2 1 Hb 140 140 110 110 5 Ht 0.41 0.43 0.36 0.33 2 MCV 93 85 75 102 3 MCH 31.8 28 22.9 34.4 Red cell membrane from Essential Haematology, AV Hoffbrand, et al. 4e 19 Red Cell Aging 20 Red Cells - transport function ▪ Oxygen uptake in lungs ▪ Changes with age ~450 km circulation ▪ saturation >95% occurs by ~1/3 distance travelled in alveolar capillaries ▪ Loss of membrane lipid ▪ Loss of Decay acceleration factor (CD55) & CD59 ► (Complement inactivation) ▪ Loss of CD47 – inhibits phagocytic clearance ▪ Some enzymes decrease ▪ Oxidative damage to lipids & proteins ▪ Cross-linking of structural components ▪ CO2 transport from tissues ▪ CO2 enters red cells ► rapidly converted to H2CO3 – carbonic anhydrase ► HCO3-, diffuses out, H+ bound by Hb - minimises pH shift ! ▪ Nitric oxide – transport & reserve for BP control ▪ Structural anatomy critical - ▪ End result: ► Increased rigidity and surface changes ▪ Reduced ability to repair oxidative damage ▪ Leading to phagocytosis by macrophages ► splenic cords, marrow & liver sinusoids 21 Normal haemoglobins ▪ large alveolar area ▪ thin layer for diffusion ► epithelial cell - ground substance - endothelial cell ▪ red cells squeeze through capillaries (<8 um diam.) 22 O2 uptake & release by Haemoglobin Bohr effect ▪ Tetrapeptide - Mr 68,000 ❖ Sigmoid curves – affinity changes as Hb binds O2 ▪ Adult Hb: Hb A: α2β2 ▪ 4 haem groups ▪ 4 oxygen (O2) molecules carried ❖ pH lower in tissues – shifts dissoc curve to right. ▪ HbA2 ▪ HbF ! ! ❖ CO2 enters RBC: HCO3released, globin binds H+ ▪ Minor haemoglobins in adult: α2δ2 1.5-3.2% α 2γ 2 0.5-0.8% Relaxed high affinity Tense low affinity 23 Mean oxygen tension Mean oxygen saturation Arterial 95 mm Hg 95% Venous 40 mm Hg 70% 24 Synthesis of Hb: critical nutrients Iron Metabolism ▪ Iron - an essential nutrient, but two key issues: ► Iron ►For ► Folate & Vit. B12 ►Required ! ► ▪ A toxic metal that must be kept chelated / sequestered in ferritin ▪ ability to absorb iron is restricted because excess iron cannot be excreted haem production ► Hard for thymidine synthesis ▪ Iron deficiency is the commonest cause of anaemia Vit B6 - pyridoxine ► ► to absorb excrete ► Cannot Required for haem synthesis But deficiency is very rare (alcoholics, and isoniazid therapy) ▪ Causes differ in different cultures and socio-economic environments 25 26 Iron distribution in the body Dietary iron ▪ Iron in food - DISTRIBUTION OF IRON IN THE BODY COMPARTMENTS - AVERAGE ADULT (mg) Male Female 2400 mg 1700 mg 65% Stores: ferritin, haemosiderin 1000 300 30% variable Myoglobin 150 120 3.5% Haem enzymes 20 15 0.5% Transferrin 4 3 0.1% Circulating red cells ▪ ferric – hydroxides – limited absorption ▪ ferric - protein complexes – moderately absorbed ▪ haem-protein complexes - most readily absorbed % Total iron ▪ Western non-vegetarian diet ▪ ~10-15 mg iron ▪ ~5-10% absorbed ▪ increased to 20-30% if iron deficient or pregnant ▪ Vegetarian diet ▪ Lower iron availability, 2-5% absorbed 27 28 The mucosal (enterocyte) iron block Iron absorption Iron absorption is regulated at two levels ▪ Mucosal block exists: ► Fe *** DMT-1 Divalent metal transporter (Fe+ +, Ca++, Zn++) DMT expression is regulated by iron status Fe++ Fe+++ Stored in ferritin HCP-1 transporter Haem absorption in duodenum and proximal jejunum iron – controlled: DMT-1 ► enterocytes take up Fe++ ► Haem uptake – HCP-1 ► Fe+++ absorption (minor) ▪ Fe stored in apoferritin (ferritin) - Fe+++ ▪ Fe released to plasma transferrin – controlled: ferroportin ► Hepcidin regulates ferroportin concentration (inversely) *** Ferroportin Expression is increased by low levels of Hepcidin ▪ Enterocytes shed into gut lumen at end of lifespan - ferritin iron lost ▪ Fe not absorbed in distal small bowel: insoluble at alkaline pH ► Enteric Transferrin Iron uptake is restricted by ionic state and release to transferrin is strictly controlled. A specific pathway exists for haem absorption – facilitated uptake of iron. transport rate determined when enteric stem cells divide ► Absorption 29 cell mechanisms: iron regulatory proteins bind to iron response elements of mRNA ► high Fe level increases translation of ferritin mRNA ► low Fe status increases translation of mRNA for Tf & ferroportin 30 Basics of iron handling ► Iron absorption ► Iron status of individual ► Iron transport in plasma The Iron Cycle Daily loss ~1(-1.5) mg/d Duodenum - food ▪ Some foods promote and others reduce iron absorption urine, faeces, nails, hair, skin Iron not absorbed from food: 70-95% ▪ Absorption increased if iron stores low ~1(-1.5) mg/d absorbed ▪ Transferrin – up to 2 Fe++ atoms per molecule ▪ Saturation: 15-50% (about 15-50% of the available bi ► Iron storage ► Assess iron status of patients before all major elective surgery Tissue iron liver, kidney, muscle, etc, ~0.15g) Transferrin (~1/3 saturated with Fe) ! ! Plasma ~4mg ▪ Ferritin (soluble) ▪ Haemosiderin (particulate) Bone marrow (erythroblasts ~20mg iron/d) ` Macrophages (~0.5-1.5g) Erythrocytes circulating haemoglobin Haemorrhage 31 (menstrual loss ~0.5-1mg/d) - Another way of viewing Iron status modified from Essential Haematology, Hoffbrand, Pettit & 32 Moss, 4th ed Laboratory Tests for Iron Status – Modified from Essential Haematology, Hoffbrand, Pettit & Moss, 4th ed Daily loss ~1mg/d Duodenum ~1(-1.5) mg/d urine, faeces, nails, hair, skin Transport iron ! Transferrin Iron pools storage iron Transferrin red cell & tissue iron normal iron overload - 33 Laboratory Tests for Iron Status – urine, faeces, nails, hair, skin ! Transferrin (~1/3 saturated) ! ► Blood Tissue iron (menstrual loss ~0.5-1 mg/d) (menstrual loss ~0.5-1mg/d) 34 screen – helpful screening test: MCV, MCH, Hb ► Serum iron, transferrin and % saturation of transferrin ▪ Not quite as reliable as ferritin for iron deficiency ▪ Helpful for assessing iron overload ! ! Macrophages (~0.5-1.5 g) (Ferritin) Erythrocytes ! ! circulating haemoglobin Haemorrhage Haemorrhage Serum ferritin leakage from stores ▪ Best overall test but elevated if inflammation or hepatocellular injury present liver, kidney, muscle, etc, ~0.15 g) Plasma iron ~4mg Transferrin receptors - free in plasma Erythrocytes ! ! circulating haemoglobin ► Ferritin ! !! Bone marrow (erythroblasts ~20 mg iron/d) Macrophages (~0.5-1.5g) (Ferritin) Laboratory tests for iron status Daily loss ~1 mg/d Transport iron Plasma iron ~4mg Transferrin receptors - free in plasma Modified from Essential Haematology, Hoffbrand, Pettit & Moss, 4th ed Duodenum ~1(-1.5) mg/d ! !! Zinc protoporphyrin Cheap, not as specific as ferritin (in the absence of inflammation) Bone marrow (erythroblasts ~20mg iron/d) low iron iron iron stores: deficiency deficiency incipient anaemia iron deficiency progressive iron depletion (~1/3 saturated) ! Tissue iron liver, kidney, muscle, etc, ~0.15g) Serum ferritin leakage from stores ► 35 Complex cases where inflammation / necrosis is present ▪ Zinc protoporphyrin ► in iron deficiency – Zn replaces Fe in protoporphyrin ▪ Soluble Transferrin Receptors – helpful in complex cases where there is a need to confirm a diagnosis of iron deficiency ► Derived mainly from erythroblasts 36 Iron status in health Iron storage - Daily iron losses – ▪ Ferritin Vary with age, sex, pregnancy and frequency of blood donation ▪ Outer protein shell - apoferritin soluble by all cells to store excess iron ► Largest amounts in macrophages and liver cells Urine, sweat Menstr. Pregnancy Growth & faeces ► Water ► Produced Adult male ▪ Iron (Fe+++)-phosphate-hydroxide core ▪ Varying saturation with iron ► Up to 5000 iron atoms per molecule of ferritin 0.5-1 Post menopause female 0.5-1 Menstruating female 0.5-1 Pregnant female 0.5-1 Children (average) ▪ Trace amounts of ferritin leak out of cells: plasma ferritin Female 12-15 yrs ▪ Haemosiderin Adult blood donor: ▪ aggregated ferritin, insoluble, visible in light microscope – 200 - 250 mg iron in 500 mL blood tract ► Uterus - menstruation ► Trauma ► insufficient to cope with losses and growth 1.1 0.6 1.6-2.6 0.6 mg/day 2 1.2 3 1.8 4 2.4 platelet count may be increased - bleeding / inflammation In this film – a small number of red cells are well-filled with haemoglobin suggesting that this patient is now receiving iron replacement treatment. ►Malabsorption ► Coeliac disease are uncommon 39 ▪ Oral iron for up to 3-6 months film - dimorphic population of red cells iron to replace Hb and stores ▪ Anaemia of chronic disease ► sufficient ▪ Parenteral: Fe polymaltose ► Rapid, ▪ chronic infection, inflammation or malignancy ► mild or moderate anaemia microcytosis ► Ferritin is normal or increased ► Serum iron and TIBC are decreased ► Serum iron saturation is usually decreased ► Mediated by Hepcidin: binds to Ferroportin, causing removal & internalisation of Ferroportin quantified, useful where heavy iron loss is ongoing ▪ Prophylaxis: treatment to prevent Fe deficiency ► Pregnancy ► Premature 40 Other anaemias: normocytic (normochromic) but with a tendency to hypochromic microcytic features Treatment of iron deficiency ► blood 38 some pencil cells ►Diet ► Others additional 0.6 anisocytosis and some bizarre cells ►Growth neonate, childhood 0.5-1 1.5-3 microcytic hypochromic rbc ► Gastrointestinal ► pregnancy, 1-2 1-2 Iron deficiency – blood screen Causes of iron deficiency ►Bleeding 0.5-1 1 donation / year - 37 0.5-1 0.5-1 0.5 0.5-1 Avge Total ► Often – where testing shows a need infants ▪ Causes of failure of treatment: Lab monitoring of treatment is essential ► bleeding ► not taking iron ► wrong diagnosis ► malabsorption ▪ Oral iron has no benefit - not iron deficient ► Problem ► Hepatic 41 - reduced iron release from macrophages hepcidin increased – iron absorption reduced 42 Other microcytic anaemias Examples: Inflammation & infection ▪ Thalassaemias ▪ Genetic defect in globin production (b or a) ► Variable severity anaemia ► Serum iron normal or raised ► Serum ferritin and iron stores usually increased ► Common in parts of Mediterranean, Africa & Asia ▪ Surgery ► Microcytic ▪ acute fall in serum iron level and serum transferrin ▪ a transient effect in acute inflammation ▪ but persistent in chronic inflammation The film shows a mild form of beta thalassaemia in which there is microcytosis with pencil cells and mild anisochromia. Compare the red cell size with the nucleus of the small lymphocyte. Hb 121, MCV 60, MCH 19.0 ▪ Iron is unavailable to developing red cells 43 Other hypochromic anaemias Metabolism in the Red Cell - 1 ▪ Myelodysplasia (can have microcytic cells but macrocytic overall) ▪ No mitochondria in mature red cells ▪ Energy is derived from - ▪ Premalignant disorder ► Abnormal clone haemopoietic stem cell clone ► Refractory anaemia often with ringed sideroblasts ► Defect in haem synthesis – iron persists as a ring of granules around erythroblast nucleus ► Mixture of hypochromic and hyperchromic cells – usually macrocytic ► Raised ferritin, raised serum iron, and low TIBC ► Leucocyte and platelet dysfunction may be present ►Glycolysis ▪ For each molecule of glucose catabolised: ► Used: ► Produced: ►Hexose ▪ Lead poisoning ► mild cells show basophilic stippling anaemia Fe+++ Methaem reduced to Fe ++ Haem ! ADP ! ATP ! ADP ! NAD ! NADH ! 46 Metabolism - 2 Glucose ! G-6-P Fructose-6-P ! Fructose-1,6-DP ! Glyceraldehyde-3-P ! 1,3-BPG 3-BPG Hexose monophosphate shunt monophosphate shunt 45 Major enzyme pathways in red cells ATP 2 molecules ATP 4 molecules ATP 2 molecules NADH (2 molecules lactate) ▪ 2 molecules NADPH produced / molecule glucose ▪ Defect in haem and globin synthesis ► Red 44 NADP NADPH Redox reactions H2O2 cleared Dihydroxyacetone-P ▪ ATP required for ►ion pumps ▪ High osmotic pressure ▪ Cell volume, shape, flexibility ▪ Cell membrane ATPases (Na-K) & Ca: 1 molecule of ATP used to pump 3 Na+ ions out & 2 K+ ions in 2,3-DPG ! !! ! Pyruvate 2x ATP ! 2x ATP Lactate 47 48 Megaloblastic anaemia Metabolism - 3 ▪ NADH ►In the normal red cell, haemoglobin Hb(Fe++) is slowly oxidised to Methaemoglobin - Hb(Fe+++) ►MetHb + NADH -> Hb to permit (Methaemoglobin reductase) oxygen transport ▪ NADPH ►Needed to reverse oxidative damage to the red cell membrane and proteins ▪ Maintains a pool of reduced Glutathione 49 Haemolytic anaemia ► Increased 50 Haemolytic anaemia: laboratory features rate of rbc destruction ► Increased (normocytic or macrocytic – reticulocytes are big) serum unconjugated bilirubin ► ↓ serum haptoglobin ► ↑ LDH but non-specific lifespan <120 days ►Extravascular haemolysis (spleen usually) ►Intravascular haemolysis uncommon as a dominant feature ► Clinical rbc breakdown ► anaemia ►RBC ►↑ ! ► Increased rbc production ► Polychromasia in blood film – immature reticulocyte fraction increased ► marrow erythroid hyperplasia features ► reticulocytosis ►Anaemia, jaundice, splenomegaly gallstones - if chronic ►“Aplastic” crises: parvovirus, folate deficiency ►Pigment ► ► ! Damaged rbc – red cell fragments (in some forms of microvascular or mechanical haemolysis) ! (Folate deficiency) 51 Haemolytic anaemia: classification Intravascular haemolysis ► mismatched ► G6PD ► hereditary ▪ membrane e.g. hereditary spherocytosis & ovalocytosis ▪ metabolic e.g. G6PD deficiency ▪ haemoglobinopathy e.g. thalassaemia, sickle cell anaemia ► acquired ▪ ▪ ▪ ▪ ▪ 52 immune (transfusion, fetal; autoimmune) rbc fragmentation infection chemical, drug secondary (liver, renal – usually milder disease) blood transfusion deficiency (splenic destruction) ▪ acute episodes of oxidative stress ► rbc fragmentation syndromes (microangiopathic) haemolytic anaemia (splenic destruction) ► immune ▪ some autoimmune ▪ some drug-induced ► infection (C. perfringens, et al) Hb (splenic destruction) ► March haemoglobinuria (rare) ► Paroxysmal Nocturnal Haemoglobinuria – cells susceptible to complement-mediated lysis ► unstable 53 54 Microangiopathic haemolysis Thrombotic microangiopathy Intravascular haemolysis Hbaemia Fragmented red cells: normally < 1% of red cells methaemalbumin haemoglobinuria urinary haemosiderin 55 56 Hereditary spherocytosis - Hereditary spherocytosis inherited haemolytic membrane disorder ► Polychromatic red cell Commonest inherited haemolytic anaemia in Europeans ▪ Autosomal dominant ▪ Cytoskeletal protein defect – can’t hold onto membrane ► spectrin Thrombotic microangiopathy – fibrin production following damage to endothelium Spherocyte defect most common ▪ Loss of cell membrane ► Reduced volume results in RBC becoming spherocytes ▪ Loss of cell flexibility ▪ Trapped and destroyed in splenic cords 57 58 Enzyme abnormality – oxidative haemolysis Membrane structure Glucose 6 phosphate dehydrogenase deficiency ► G6PD: required to protect rbc from oxidative damage Spectrin lattice intersecting with actin and band 4.1 proteins to form a loose hexagonal lattice. ! Oxidative radicals neutralised The lattice structure is defective in hereditary spherocytosis and elliptocytosis. glucose reduced glutathione ! Caused by several different membrane protein abnormalities, resulting in loss of lipid from the membrane and increased Na+ leakage. NADP G6P oxidised glutathione membrane damage! Heinz bodies NADPH G6PD 6PG NADP NADPH 59 lactate pentose 5-P 60 G6PD deficiency - blood film during a severe haemolytic crisis G6PD deficiency ► ► ► X-linked disorder Mediterranean, Middle East, SE Asia, West Africa Intravascular haemolysis in the face of oxidant stress 1. Severe anaemia ▪ Fava beans: precipitate haemolysis – some cases ▪ Acute infection: oxidative stress - neutrophils ▪ Oxidative drugs: antimalarials, sulphonamides, etc •Sphered and retracted cells •Pale ‘blister’ zone on one side of some cells (see Heinz body below) •Reticulocytes increased (marrow response) and occasional erythroblasts may be seen 1 2. Special (reticulocyte) stain: Heinz bodies: blue stained mass of oxidised, denatured and aggregated haemoglobin attached to inside of red cell membrane. 2 Precipitated haemoglobin (Heinz bodies) getting stuck in the spleen Reticulocyte 61 Sickle cell anaemia – haemolysis 62 Sickle cell anaemia α2βs2 ► Single amino acid substitution in β globin chain (6Glu→Val) ► Homozygotes have severe HA with crises ► HbS crystallises under low oxygen tension ► HbS ▪ infection, surgery ► Importance of dehydration (sickle cell trait) only symptomatic under severe anoxia (anaesthesia, pregnancy) ► Heterozygotes ►Vascular obstruction ►Infection 63 64 Thalassaemia Sickle cell anaemia Beta globin gene Beta thal X X 65 Alpha globin gene Alpha thal 66 Thalassaemia Normal Hb production ► genetic defect in production of either α or β globin chains leading to: ▪ Microcytic anaemia ►plus shortened rbc survival and ineffective erythropoiesis ►β-thalassaemia - α-globin/heme aggregates ►α-thalassaemia - β4 tetramers precipitates α HbA β HbF γ % total globin synthesis Early death of developing red cells = ineffective erythropoiesis HbA α2β2 HbF α2γ2 HbA2 α2δ2 ε, ζ 67 β Thalassaemia 6 δ Hb A2 birth weeks 30 68 β thalassaemia homozygote ► Usually point mutation ► Heterozygous state: trait (asymptomatic) ► Homozygous or compound heterozygous: thalassaemia major ▪ Symptomatic after six months of age ▪ Transfusion dependent ▪ Skeletal abnormalities ▪ Iron overload despite chelation therapy 69 70 β thalassaemia homozygote Haematopoiesis Massive expansion of bone marrow to compensate – bony deformities 71 72 β thalassaemia homozygote α Thalassaemia ► Four α globin genes: 2 from each parent ► Deletion of one or more α globin genes: ▪ 1 gene deletion: silent carrier ▪ 2 gene deletion trait: – mild ►but beware of risk to offspring if partner also has 2 gene deletion ▪ 3 gene deletion: HbH disease (β chain tetramers) ►severe anaemia ± transfusion dependency ▪ 4 gene deletion: hydrops fetalis syndrome ►lethal in utero 73 74 Haemoglobinopathies Alpha thalassaemia 75 76 Examples of acquired haemolytic anaemias malaria burns autoantibody Immune haemolysis warm type (IgG) ► ► ► Red cells parasitised haemolysis occurs after parasite grows to maturity Thermal injury to red cells microspherocytes produced: cleared rapidly Opsonisation of red cells by macrophages in spleen. Spherocytes, reticulocytosis and erythroblasts in blood 77 idiopathic secondary ▪ SLE etc ▪ CLL, lymphomas drugs IgG (+IgM, IgA, C3b/d) cold type (IgM) ► ► ► idiopathic secondary ▪ Mycoplasma ▪ infectious mononucleosis ▪ lymphoma IgM + peripheral agglutination & slow Complement activation Direct Coomb’s test = Direct Antiglobulin Test (DAT)! detects IgG or Complement on rbc’s: anti-IgG + anti-C3b/d 78 To answer the question about direct and indirect antiglobulin test from yesterday Immune haemolysis: drug induced autoantibody Anaemia Spherocytes Polychromasia Reticulocytes raised Bilirubin increased 79 Acute lymphoblastic leukaemia – immature lymphoids/ lymphobasts Acute myeloid leukaemia – immature myelobasts Lymphomas and mature lymphocyte cancers (CLL, myeloma etc) } } Myeloproliferative neoplasms – too many mature forms 80 Acute leukaemia Lymphoid series (lymphocytes) ► accumulation of malignant blast (primitive) cells, that fail to differentiate. ► replace normal marrow and cause bone marrow failure ► clonal proliferation normal cells Myeloid series Also myelodysplastic syndrome – odd and ineffective myeloid cells Acute leukaemia Marrow population (log scale) symptom threshold somatic mutation ∗ tumour cells 82 time Acute lymphoblastic leukaemia 83 84 Acute myeloid leukaemia (AML) ► all age groups ► commonest leukaemia in adults ► Usually de novo but ► may follow – WHO: ALL ▪ myelodysplasia, ▪ myeloproliferative disease: o Polycythaemia, Chr. Myeloid Leuk., Myelofibrosis ► blast WHO: Burkitt leukaemia cells of some types have Auer rods 85 86 Acute leukaemia: investigations ► Peripheral ! ► Bone Treatment Concepts blood screen marrow aspirate ! ► Immunological classification by flow cytometry ▪ Immunophenotyping – cell surface marker molecules ► Cytogenetics / Molecular genetics ▪ Disease type characterised – WHO classification 87 AML classification - AML – other variant types AML, not otherwise categorised ► AML WHO 88 FAB minimally differentiated M0 ► AML without maturation M1 ► AML with maturation M2 ► Acute promyelocytic leukaemia M3 ► Acute myelomonocytic leukaemia M4 ► Acute monoblastic/monocytic leukaemia M5 ► Acute erythroid leukaemia M6 ► Acute megakaryoblastic leukaemia M7 ► …. ► AML - with recurrent cytogenetic abnormalities, eg ► AML with t(8;21) with t(9;22) ► AML with t(16;16) or inv(16) ► AML with 11q23 abnormalities ► AML with t(15;17) ► AML ▪ Acute Promyelocytic Leukaemia ► AML - with multilineage dysplasia ► AML - treatment related ► abnormal 89 ► Alkylating maturation in at least 2 cell lines agents, Topoisomerase inhibitors 90 Auer rods in AML (M2) Acute myeloid leukaemia ► AML M3 acute promyelocytic leukaemia ► AML M4 (myelomonocytic) and M5 (monocytic) ▪ associated with chronic DIC ▪ t(15;17) RAR-PML gene sites ▪ tissue infiltration ► Remission can be achieved in a majority of patients, but it is generally short-lived ► Long term survival 20% ► Bone marrow transplantation (either autologous or allogeneic) improves the prognosis 91 92 CML: t(9;22) Chronic myelogenous leukaemia (CML) ► Philadelphia chromosome the translocation results in a fusion between BCR (chr 22) and abl (Abelson oncogene) (chr 9) ► middle aged ► massive expansion of granulocyte mass ▪ Most of the leukaemic cells are differentiated ▪ hypermetabolism ► sweats, ! ! ! weight loss, anorexia ▪ splenomegaly ▪ marrow failure ►New fusion gene that codes for a novel tyrosine kinase I XI to Glivec (imatinib): blocks hybrid ▪ Responsive BCR-abl tyrosine kinase ► undergo a terminal transformation to AML ► mean survival 3-4 years – with conventional cytotoxic treatment ►Cells differentiate and die further mutations later result in drug resistance (not very often) ►But, 93 LYMPHOID CANCERS Myelodysplasia ► neoplastic / pre-neoplastic marrow ► abnormalities of all three cell lines 94 & blood condition Chronic lymphocytic leukaemia ▪ stem cell disorder ▪ most arise de novo, some occur in patients with previous chemotherapy ► ineffective cell maturation despite increased marrow cellularity ► cytopenias, ▪ anaemia with macrocytosis usually ▪ may have thrombocytopenia + impaired platelet function ▪ may have neutropenia + impaired neutrophil function ► tendency to progress to AML (~ 30%) 95 96 Multiple (plasma cell) myeloma Chronic lymphocytic leukaemia 97 Multiple (plasma cell) myeloma Hodgkin lymphoma ▪ Anaemia ▪ Bone erosion & fractures (lytic lesions) ►stimulate osteoclasts (IL-6) immunoglobulin in most but ~20% only λ or κ light chains (light chain myeloma) ► presence ►Monoclonal ▪ Reduced levels of polyclonal Ig – bacterial infection risk ▪ hypercalcaemia, ▪ renal failure – tubule obstruction ►Ca + light chains + urinary mucoprotein: precipitates ▪ Therapy ►Cytotoxic 98 therapy / autologous BMT in younger age group of Reed Sternberg cells ▪ bi-or multi-nucleate ▪ owl’s eye appearance ▪ malignant cell is a B cell ► often present in single node area ► disseminates to others ► splenomegaly 50% ► prognosis and treatment depends on stage ▪ > 90% survival stage I-II 99 100 Staging for Hodgkin’s disease Hodgkin lymphoma ► Classic mass presentation = young adults with mediastinal ► Usually nodular sclerosing HD ► Constitutional symptoms ► Fever, night sweats, weight loss, pruritus ► Reduced T cell immunity: viral & fungal inf. ► antibody responses preserved until late ► Early Stage I Stage II Stage III stage disease: excellent prognosis ► Advanced disease > 50% 5 year survival ! ► Risk of chemotherapy-induced myelodysplasia & AML Stage IV 101 102 Non-Hodgkin lymphoma ► ► ► ► ► ► ► Hodgkin lymphoma Majority arise from germinal follicle centre cells Architecture may be either follicular or diffuse 90% are B cell derived More malignant forms have larger cells with high rates of proliferation and diffuse infiltration of node Many many types: Diffuse lymphoma most common, then follicular lymphoma. Burkitt lymphoma Nodes at presentation Spread Mesenteric nodes, Waldeyer ring ▪ African children, jaw mass, EBV, Adult T cell leukaemia / lymphoma ▪ HTLV-1, CD4+ T cells ▪ Caribbean and Southern Japan Extranodal involvement 103 single group, axial contiguous node areas Non-Hodgkin lymphoma multiple areas non-contiguous rare more common uncommon common 104