Cellular and Transfusion Science Haematology Lecture 1 PDF

Cellular and Transfusion Science AP0509 2024-25 Haematology Dr Antony Antoniou [email protected]. uk 1 Cellular and Transfusion Science AP0509 2024-25 Haematology Haematopoiesis Erythropoiesis Anaemia Classificati...

Cellular and Transfusion Science AP0509 2024-25 Haematology Dr Antony Antoniou [email protected]. uk 1 Cellular and Transfusion Science AP0509 2024-25 Haematology Haematopoiesis Erythropoiesis Anaemia Classifications Anaemias Haemoglobin Synthesis Cellular and Transfusion Science AP0509 2024-25 Haematology Lecture 1 Part 1 Haematopoiesis Erythropoiesis Anaemia Classifications Anaemias  Haematopoiesis Aims and Objectives Haematopoiesis; principles of blood cell type development (Hoffbrand’s Essential Haematology Chpt 1) Stem cell hypothesis; priniciples of self-renewal and commitment (Hoffbrand’s Essential Haematology Chpt 1) Erythropoiesis; development of red blood cells (Hoffbrand’s Essential Haematology Chpt 2)  5 Table 23-1 Molecular Biology of the Cell (© Garland Science 2008)  6 Wrights stain Figure 23-37e Molecular Biology of the Cell (© Garland Science 2008)  7 Stem Cell Theory of Haematopoiesis  Cell Differentiation 8 Definitions TOTIPOTENT; form all cells including extraembryonic and placental cells PLURIPOTENT; give rise to all cell types MULTIPOTENT; give rise to more than one cell type but limited All blood cells have common ancestral cell – MULTIPOTENT STEM CELL (MSC) MSC has the option of differentiating to; – Colony Forming Unit-Granulocyte, Erythrocyte, Monocyte, Megaokaryocyte (CFU-GEMM); myeloid cell line – late RBC’s, platelets, granulocytes and monocytes – Lymphoid stem cell (lymphoid cell line – later lymphocytes and natural killer cells)  9 Stem Cell Theory of Haematopoiesis All cells derived from a pool of stem cells that are self-renewing Pluripotential & multipotential stem cells give rise to committed stem cells for each cell line Committed stem cells have receptors for specific growth factors Respond to stimulation by division & maturation (precursor cell stages) into end-stage cells  Characteristics of 10 Hematopoietic Stem Cells (HSC) Stem cells defined as capable of both self renewal and multi-lineage differentiation HSC are multi-potent stem cells that occur at a frequency of 1:5000 in bone marrow HSC can be defined by: Function: the capacity to give rise to non-self renewing populations that generate multiple terminally-differentiated cell types Surface phenotype: c-kit+Sca-1+Lin-Thy1lo  11 Cell Fate Decisions During Hematopoiesis LONG TERM -indefinite self renewal SHORT TERM -definitive self renewal MULTIPOTENT PROGENITOR COMMON LYMPHOID PROGENITOR COMMON MYELOID PROGENITOR KEY; MEP- MEGAKARYOTIC/ERYTHROID PROGENITOR, Passegue et al. (2003) GMP-MYELOMONOCYTIC PROGENITOR, DC-DENDRITIC CELL NK-NATURAL KILLER CELL PNAS 100: 11842-11849  12 WHERE? Self renewal Stem cell - embryonic; inner cell mass of blastocysts - Adult; most tissues e.g.bone marrow Committment  13 Development of Blood Cells 3 wk : formation of blood islands from yolk sac 6 wk : liver becomes hematopoietic organ 6-8 wk : spleen (until 8th month) ~20wk : bone marrow (life-long)  14 Stem Cell Niches within the Bone Marrow Stem cell niche is a specific site (microenvironment) in adult tissues where stem cells reside and undergo renewal and differentiation Bone marrow harbours two distinct hematopoietic niches: (1) Osteoblastic niche at the endosteal surface (2) Vascular niche involving sinusoidal blood vessels These niches have distinct yet complementary functions in helping to balance self- renewal with differentiation Osteoblastic niche maintains quiescence and harbours the Long Term-HSC Vascular niche supports proliferation, differentiation and mobilization (transendothelial migration) of Short Term-HSC to the blood stream in response to physiological demands and act as back up outside the BM for HSC during times of BM stress  15 The Osteoblastic and Vascular Niche Yin et al. (2006) J Clin Invest 116:1195-1201  16 HOW are blood cells generated? Self renewal Stem cell - embryonic; inner cell mass of blastocysts - Adult; most tissues e.g.bone marrow Committment  17 Intrinsic versus Extrinsic Factors Controlling Hematopoiesis Cell fate determination is governed by interactions between extrinsic and intrinsic factors Soluble growth factors (extrinsic) Transcription factors (intrinsic)  18 Transcription Factors Specifying Cell Fate Decisions Runx1 Runt related transcription factor family -required for normal hematopoiesis Important regulators of Ikaros immune system development Essential for erthryroid and GATA-1 Pax5, E2A, EBF platelet forming cells Notch-1 GATA-3 T cell development  19 Hematopoiesis Proliferative differentiation potential  20 Summary Hematopoietic stem cells give rise to cells of the blood system Hematopoiesis site changes during development HSC renewal and commitment occurs within distinct microenvironments of the bone marrow Extracellular signals & internal molecular programmes govern cell fate  1 Cellular and Transfusion Science AP0509 202-23 Haematology Lecture 1 Part 2 Haematopoiesis Erythropoiesis Anaemia Classifications Anaemias  2 Erythropoiesis (generation of red blood cells) 5-7 days Erythropoiesis (generation of red blood cells) 3 Proerythroblast First cell committed to RBC basophilic erythroblast nucleus becomes smaller cytoplasm becomes more basophilic due to the presence of ribosome polychromatophilic erythroblast produce more haemoglobin cytoplasm starts to take up both basophilic and eosinophilic stains orthochromatophilic erythroblast extrudes nucleus Reticulocyte cytoplasm containing reticular networks of polyribosomes Enters circulation Figure 23-44 Molecular Biology of the Cell (© Garland Science 2008)  Visualisation 4 1 2 Normoblast Basophilic normoblast 3 4 Polychromatophilic Normoblast Orthrochromatophilic Normoblast  RETICULOCYTES 5 Young red blood cell; still have small amounts of Reticulocyte RNA tend to stain more blue than mature RBC’s on Wright stain (polychromatophilic) Slightly larger than mature RBC Undergo removal of RNA on passing through spleen in 1st day of life Important marker of RBC production Reticulocyte count= Retic % x RBC Count eg 0.01 x 5,000,000 = 50,000 Normal up to 100,000  6 Erythropoiesis-where? HSC; Haematopoeitic stem cells, BFU-E; burst forming unit-erythroid, CFU-E; colon forming unit-erythroid  7 Intrinsic factors-transcription factors Extrinsic factors-soluble factors Extrinsic Factors; Erythropoietin 8  9 Erythropoietin  10 Erythropoietin (EPO) Epo Receptor (R) Phosphorylate STATs Survival Differentiation EPO receptor; Proliferation haematopoietin receptor superfamily dimerize after binding their ligand.  RED BLOOD CELLS 11 Normal - Anucleate, highly flexible biconcave discs, 80-100 femtoliters in volume Flexibility essential for passage through capillaries Major roles - carriers of oxygen to & carbon dioxide away from cells  12 Synthesit Sep 21,2021 (www.syntheist.ee)  13 RED BLOOD CELL METABOLISM Red blood cells function without a nucleus and mitochondria Only nucleated RBC; normoblasts (RBC precursor) and megaloblasts which appear in megaloblastic anaemia No nucleus; enhances flexibility, restricts size, increasing O2 carrying capacity Reduced life span  RED BLOOD CELL METABOLISM Pentose phosphate pathway - Generates reduced NAD i.e. NADPH - NADPH generates reduced glutathione (anti-oxidant) - Generation of reduced glutathione stimulates glucose metabolism Methemoglobin reductase pathway - Maintains iron in Fe2+ state Luebering-Rapaport Bypass - 2,3-DPG regulates O2 carrying capacity and release Lactic acid fermentation - Produces NAD+ and ATP n Wijk and van Solinge Blood 2005, 106 p4034-42 14 own Lab Medicine 1996, 27 p329-33  15 RED BLOOD CELL METABOLISM No mitochondria; do not use up oxygen RBC make ATP by; EMBDEN MEYERHOF PATHWAY - glycolysis of glucose to pyruvate forming Adenosine TriPhosphate (ATP) and Nicotinamide Adenine Dinucleotide (NADH) - lactic acid fermentation on pyruvate forming ATP, NAD+, lactate Pentose phosphate pathway (hexose monophosphate shunt) -important in generating NADPH, -glucose-6-phosphate dehydrogenase important regulatory enzyme, -help prevent oxidative stress -to reduce the oxidated form of glutathione -keep haemaglobin in ferrous state (Fe2+ allow O2 binding)  16 Summary Red blood cells formed during Erythropoiesis Erythropoiesis composed of 5 distinct stages Governed by intrinsic and extrinsic factors EPO important extrinsic factor Red Blood Cells short lived but equipped to function without mitochondria and nucleus Maintain biochemical pathways for energy generation, protection and optimal function  1 Cellular and Transfusion Science AP0509 2022-23 Haematology Lecture 1 Part 3 Haematopoiesis Erythropoiesis Anaemia Classifications Anaemias  2 ANAEMIAS AIMS Classification of anaemia Characteristics and diagnostics  3 Disturbance of ↓ Prolifn/Diffn DNA synthesis Erythroid precursor Disturbance of Hb synthesis ↓ Prolifn/Diffn Unknown/ Of HSC ↓Production Multiple mechanisms ANAEMIA Mechanical Membrane ↑Destruction Enzyme Defect ↑ Loss Blood loss Defect Chemical/ Physical Intrinsic Extrinsic Abnormality Abnormality Infection Globin Abnormalities Hypersplenism Antibody Mediated  Anaemia 4 Definition by WHO haemaglobin < 13g/dL (men), 100 fl and a normal MCHC. Cells are much larger but the cellular [Hb] is normal.  Megaloblastic anaemias  certain haemolytic anaemias. Microcytic (hypochromic) (MH) anaemias:  MCV of < 80 fl and an MCHC of < 30 g/dl.  Iron deficiency anaemia (most common)  Sideroblastic anaemias (impaired haem synthesis)  Thalassaemia syndromes (impaired globin synthesis) Anaemia due to haemorrhage Acute (extrinsic): Immediately after acute blood loss, [Hb] will be normal, due to loss of red cell mass and plasma volume in the same proportion. – 24 hrs later compensatory mechanisms take effect. – Fluid enters the circulation to restore blood volume. – Haemodilution results anaemia. Start of RBC regeneration; see increase in the circulating reticulocytes Polychromasia; immature red cells may be a feature of the blood film Other parameters affected; WBC and platelet count often increased, Treatment; arrest blood loss and restore blood volume by transfusion. Chronic (extrinsic or intrinsic); Initially, anaemia develops is normochromic and normocytic. As iron stores become depleted through the generation of new red cells a microcytic/hypochromic anaemia develops ~ as in iron deficiency. Treatment; arrest bleeding and restore iron stores by giving iron. Severe cases may require additional transfusion. 7  Aplastic Anaemia (production) 8 Bone marrow does not function Red blood cell aplasia - erythroblasts; reduced or increased - Pure RBC aplasia (PRBCA) most common due to reduced erythroblasts - Congenital, most common DIAMOND-BLACKFAN ANAEMIA (congenital hypoplastic anaemia), ribosomal protein genes - Myelodysplastic syndrome-excessive fibroblast growth Acquired PRBCA Causes; Primary or secondary - infections; virus e.g. HIV, bacteria e.g. staphylococcal - Solid and haematological tumours - Autoimmune disease - Drugs and chemical Treatments; transfusions, corticosteroids, bone marrow  Decreased Production Macrocytic (megaloblastic) Anaemia Disorder of DNA synthesis, cells undergo incorrect division (thus larger) B12 and/or folate deficiency. Dietary or due to malabsorption. Investigation shows ↓Hb, with ↑MCV ANISOPOIKILOCYTOSIS Further investigation; assays for B12 and folate, + Schilling test and Intrinsic factor antibody Classical anaemia symptoms plus neurological symptoms. Other rapidly dividing cells (skin GI 9  10 Decreased Production Anaemia of chronic disease Normochromic/normocytic (i.e. normal cells in reduced numbers) Mechanism unclear; often seen in malignant disease, chronic inflammation HYPOCHROMIA, ANSIOCYTOSIS, and chronic infection. ELONGATED CELLS Only cured by treating underlying cause. Chronic Renal Failure Reduced production of erythroid precursors. Caused by lack of production of EPO (by kidneys) CRF- BURR CELLS (IRREGULAR Normal cells produced but in greatly CONTRACTION) reduced number. REDUCED NUMBERS OF CELLS  11 Overproduction of Red Blood Cells Polycythemia Vera (increase in all blood cells) Causes Unknown mutation in stem cells but also in JAK2 increase sensitivity to EPO Symptoms Headaches, dizziness, flushed complexion Increased blood viscosity Number of red blood cells increased for no other reason (e.g. cardiac or respiratory disease) Erythemia (increase in red blood cells) Phlebotomy is the most common treatment  Summary 12 Anaemias arise due to; changes in RBC production increase in loss or destruction Classified into 3 main types; Normochromic/normocytic (NN) Microcytic (hypochromic) (MH) Macrocytic (normochromic) (MN) Diagnosis; physical examination blood test RBC physical parameters and biochemistry  1 Cellular and Transfusion Science AP0509 2021-22 Haematology Lecture 1 Part 5 Haemolytic Anaemia  Haemolytic Anaemia A haemolytic anaemia is one which results from an increase in the rate of red blood cell destruction.  Normal life-span of 100-120 days can be shortened by varying degrees, extreme cases – few days  Removed by spleen when; dying/worn out (effete) or when erythrocytes are defective  Hypersplenism; many defective cells are produced.  Increased haemolysis; symptoms of anaemia, splenomegaly (enlarged spleen) due to increased workload  Anaemia results when new cells not made at rate of destruction. 2  Haemolytic Anaemia Shortened lifespan does not always lead to anaemia: Bone marrow can increase production 6-8 fold Maintains normal Hb level Marrow will exhibit hyperplasia (increase production) “Compensated Haemolytic disease” for haemolytic disorders with reticulocytosis, but no anaemia 3  Classifying Haemolytic Anaemia Intrinsic or Extrinsic? Is the problem within the RBC’s themselves (membrane, enzymes, globin) or outside (physical, chemical, mechanical, drugs, antibodies)? Intravascular or extravascular (majority are latter)? Where is the site of RBC destruction? – important for diagnosis. Acquired or inherited? 4  Intravascular or Extravascular Haemolysis Intravascular haemolysis; RBCs destroyed in circulation Release Hb in plasma Can be immune mediated e.g. blood group incompatibility Iron containing compounds in blood can cause damage Free Hb can bind haptoglobin, which can reduce haptoglobin levels Extravascular haemolysis; Premature destruction of the red blood cells in the spleen/bone marrow RBC removed by macrophages Haem breakdown in the macrophage generates bilirubin Bilirubin is released and bound onto albumin to the liver for conjugation and excretion in bile Rise in unconjugated bilirubin. Jaundice 5  Evidence of increased haemolysis Damaged RBC’s – Morphology: Microspherocytes, red cell fragments, sickle cells etc – Osmotic Fragility: Increased tendency to lyse in hypotonic saline. – Direct Coombs tests (immune-mediated haemolysis) – RBCs destroyed, releasing Hb into plasma Biochemical indicators – Bilirubin, haptoglobin, breakdown products Haemoglobinuria (high [Hb] in urine (different to Haematuria which is blood in urine) and often associated with haemolytic anaemia. Increased erythropoesis 6  Classifying Haemolytic Anaemia Acquired Inherited (extrinsic) (intrinsic) Immune Non-immune Membrane Globin Defects Defects Autoimmune HDN e.g. Spherocytosis, e.g. Sickle Incompatible Mechanical transfusion Chemical Drug induced Infection Enzyme Burns Defects Toxins e.g. G6PD deficiency 7 Intrinsic Haemolytic Anaemias 8 MEMBRANE AND ENZYME DEFECTS 9 (intrinsic)  10 RBC Membrane Defects Haemolytic anaemia; 3 main causes 1. Hereditary spherocytosis; skeletal membrane and lipid bilayer protein interactions, spherical, loss of flexibility 2. Hereditary elliptocytosis; oval and elliptoid, deficiency in protein 4.1 3. Paroxysmal nocturnal haemoglobinuria; mutation in phosphatidylinosital glycan A (PIG-A), defect in GPI, cells sensitive to complement – innate immune  RBC Enzyme Defects 11 Glucose-6-Phosphate Dehydrogenase; Removes hydrogen from glucose-6-phosphate and becomes 6-phosphogluconate - generates NADP  NADPH and GSSG + H  GSH - RBC-oxidative stress Enzyme deficiency of the membrane - Normal blood picture, except in “crises” - Crises precipitated by drugs, infection and broad beans! - RBC have shortened life-span, but mechanism unclear. Cause - G6PD gene Sex-linked inherited disorder (X chromosome) - Drug induced haemolysis e.g. primaquine, antibiotics, aspirin Treatment - Relatively self-limiting - Drugs causing condition avoided FRAGMENTS, BLISTER CELL, BITE CELLS – SPLEEN REMOVING PRECIPITATED Hb  RBC Membrane Defects Increased Destruction – Intrinsic Disorders Membrane disorders; Hereditary spherocytosis/elliptocytosis Abnormal membrane construction RBC’s are unusual shape and rigid Readily removed by spleen SPHEROCYTES – SMALLER, ROUNDER, LACK OF CENTRAL PALLOR, SOME Morphology – POLYCHROMASIA (RETCIS) spherocytes/elliptocytes, reticulocytosis, RBC’s smaller than usual. 12  RBC Globin Defects Increased Destruction – Intrinsic Disorders Globin Abnormalities e.g. Sickle Cell Defective Hb produced: caused by a single amino acid substitution on b-chain of molecule During sickle crises (infection, low O2 tension etc.) Hb becomes rigid – Cells distort (sickle) SICKLE CELLS, NUCLEATED RBC lifespan 10-12 days – Treatment with analgesia, and O2 13  14 Extrinsic Haemolytic Anaemias  Acquired non-immune 15 Increased Destruction – Extrinsic Abnormalities Mechanical HELMET CELLS, SCHISTOCYTES RBC’s damaged/destroyed by mechanical process Prosthetic heart valves, laying down of fibrin strands, marching, long-distance running! Lab. Findings – film shows fragments, anisopoikilocytosis and polychromasia MARKED CRENATION Chemical/Physical RBC’s damaged directly Burns victims – heat over 47’C crenates cells. Lead poisoning – direct toxic effect on RBC’s Damaged cells removed by spleen (haemolysis).  16 Acquired non-immune Increased Destruction – Extrinsic Abnormalities Infection RBC’s removed as spleen detects intracellular infection e.g. malaria Organism conducts life-span within the RBC INFECTION WITH P.vivax, RBC’S ENLARGED, ONE INFECTED CELL SHOWING TROPHOZOITE FORM Immune-mediated Haemolysis Antibodies react with RBC’s and cause destruction Causes include incompatible blood transfusion, autoantibodies e.g. lupus, drug reactions RBC’s may show autoagglutintation on film. AGGLUTINATION, POLYCHROMASIA Can be extravascular ANISOPOIKILOCYTOSIS  17 Summary Haemolytic anaemia results from increase in the rate of red blood cell destruction Anaemia results when new cells not made at rate of destruction Intrinsic (inherited) or Extrinsic (acquired) Intravascular (circulation) or extravascular (spleen) Acquired or inherited