Week 8

• Blood is made up of cells and liquid
• Plasma

  • Clear, straw-coloured liquid portion of blood that remains after cellular components are removed
  • 90% water
  • Transport medium for blood cells
  • Also contains salts, enzymes, antibodies, and other proteins
• Serum

  Fluid left after blood clots = plasma without fibrinogen
• Red blood cells (Erythrocytes)

  • Diameter: 6-8um
  • Lifespan: 120 days
  • Number: Male – 4.5-6.5 x 10^12/L, Female – 3.9 – 5.6 x 10^12/L
  • Function: Oxygen and carbon dioxide transport
  • 'Biconcave disc'
  • No nucleus
• Platelets

  • Diameter: 0.5-3um
  • Lifespan: 10 days
  • Number: 140 – 400 x 10^9/L
  • Function: Haemostasis
  • Contain fine granules
  • Should be dispersed
• White blood cells (Leucocytes)

  • Polymorphonuclear
  • Neutrophils
  • Eosinophils
  • Basophils
  • Mononuclear
  • Lymphocytes
  • B cell
  • T cell
  • Monocytes
• Neutrophil

  • Diameter: 12-15 um
  • Lifespan: 6-10 hrs
  • Number: 1.8-7.5 x 10^9/L
  • Function: Protection from bacteria and fungi
  • Fine granules in cytoplasm
  • Nucleus divided into 2-5 distinct lobes
• Eosinophil

  • Diameter: 12-15um
  • Lifespan: days
  • Number: 0.04-0.44 x 10^9/L
  • Function: Protection against parasites
  • Bi-lobed nucleus
  • Cytoplasm packed with large reddish-orange granules
• Basophil

  • Diameter: 12-15um
  • Lifespan: days
  • Number: 0.01-0.1 x 10^9/L
  • Function: Allergic reactions
  • Nucleus obscured by purple-black granules
• Lymphocytes

  • B cells (10-15%) and T cells (80%)
  • Diameter: 7-9 um (resting); 12-20 um (active)
  • Lifespan: Weeks or years
  • Number: 1.5-3.5 x 10^9/L
  • Function: B cells – immunoglobulin synthesis; T cells – protection against viruses
  • Round or slightly indented nucleus
  • Scanty cytoplasm
  • Small ones are circular; larger ones may have an irregular outline
• Monocyte

  • Diameter: 12-20um (largest)
  • Lifespan: 20-40 hrs
  • Number: 0.2-0.8 x 10^9/L
  • Function: Protection from bacteria and fungi
  • Irregular lobulated nucleus
  • Greyish-blue cytoplasm
  • Cytoplasm may contain vacuoles
• Venepuncture

  The process of drawing blood from a vein
• EDTA

  A type of anticoagulant used in blood tests
• Methods of investigating blood

  • Venepuncture
  • Automated full blood count
  • Blood film
  • Bone marrow biopsy
• Steps in automated full blood count

  • Whole blood in EDTA
  • Platelet count and size
  • Red cell count and size
  • Lysis of red cells
  • Flow cytometry to obtain white blood cell differential
• Coulter Principle

  A method of counting cells using electrical impedance
• Steps in Coulter Principle

  • Whole blood passed between 2 electrodes through single cell aperture
  • EI changes as a cell passes through the aperture
  • Change in impedance is proportional to cell volume, resulting in a cell count and measure of volume
• Flow cytometry

  A method of analysing cells by passing them in single file in front of a laser
• Measurements taken by flow cytometry

  • Passage of cells in single file in front of a laser
  • The degree to which the laser is interrupted forward (FSC) and obliquely (SSC) tells us the size and granularity of the cell
• CD Molecules

  Cluster of differentiation molecules, which are cell surface proteins that can differentiate one cell type from another
• Components of a blood film

  • Commonest stain in UK: May-Grunwald
  • Giemsa (MGG)
• Haemopoiesis

  Blood cell formation
• Erythropoiesis

  Red cell formation
• Myelopoiesis

  White cell formation
• Thrombopoiesis

  Platelet formation
• Site of haemopoiesis

  1. First few weeks of gestation: yolk sac

  2. From 6 weeks until 6-7 months of fetal life: liver and spleen (until 2 weeks after birth)

  3. 6-7 months onwards: bone marrow
• Site of haemopoeisis

  1. Infancy: all the bone marrow is haemopoietic

  2. Childhood: progressive fatty replacement

  3. Adult: confined to central skeleton and proximal ends of femurs and humeri

  4. 50% fat even in these areas

  5. Capable of reverting to haemopoiesis

  6. Liver and spleen can resume fetal haemopoietic role (‘extramedullary haemopoiesis’)
• Haemopoietic stem cells (HSC)

  • Asymmetric cell division allows self-renewal and pluripotency
  • Rare - 1 in every 20 million nucleated cells
  • Many are dormant, only entering the cell cycle ~every 20 weeks
  • Exact phenotype unknown: CD34+CD38- and negative for lineage markers
  • Look like a small lymphocyte
  • Located in ‘niches’: either endosteal (lined by osteoblasts) or vascular (lined by endothelium)
• Bone marrow microenvironment

  Stromal cells secrete molecules to form the extracellular matrix and growth factors for stem cell survival
• Haemopoietic progenitors

  • Lost capacity for self-renewal
  • Irreversibly committed to a specific lineage
  • Earliest recognised: colony-forming unit (CFU)
  • Lab tissue culture work: progenitor cell is identified by culture in a semi-solid medium by the type of colony they form
• Abbreviations

  • BFU: burst-forming unit; CFU: colony-forming unit; E: erythroid; Eo: eosinophil; GEMM: granulocyte, erythroid, monocyte and megakaryocyte; GM: granulocyte, monocyte; Meg: magakaryocyte; NK: natural killer
• How is lineage determined?

  1. Expression of transcription factors

  2. External signals such as growth factors
• Haemopoietic Growth Factors

  • Source: stromal cells (except erythropoietin – kidney and thrombopoietin – liver)
  • Glycoproteins that act at very low concentrations
  • Act hierarchically
  • Usually produced by many cell types
  • Usually affect more than one lineage
  • Usually active on stem/progenitor cells
• GATA-1 and FOG-1

  • Roles in erythropoietic and megakaryocytic differentiation
• External signals such as growth factors

  • Source: stromal cells (except erythropoietin – kidney and thrombopoietin – liver)
  • Glycoproteins that act at very low concentrations
  • Act hierarchically
  • Usually produced by many cell types
  • Usually affect more than one lineage
  • Usually active on stem/progenitor cells AND differentiated cells
  • Usually show synergistic or additive interactions with other growth factors
• Erythropoiesiscell forms

  Proerythroblast, Early erythroblast, Late erythroblast, Reticulocyte
• Myelopoiesis cell forms

  Myeloblast, Promyelocyte, Myelocyte, Metamyelocyte
• Thrombopoiesis

  Megakaryocytes undergo endoreduplication – replication of the nuclear genome without undergoing mitosis leading to elevated nuclear gene content and polyploidy
• Haemopoietic Growth Factors

  • Act on stromal cells: IL-1, TNF
  • Act on pluripotent stem cells: SCF, FLT3-L, VEGF
  • Act on multiple progenitor cells: IL-3, GM-CSF, IL-6, G-CSF, Thrombopoietin
  • Act on committed progenitor cells: G-CSF, M-CSF, IL-5, erythropoietin, thrombopoietin
• Erythropoietin comes from the kidney