Haemopoiesis

Haemopoiesis

1. What is it?

   • Refers to the production of all the cells of the blood
     • Can be further split into
       • Erythropoiesis (RBCs)
       • Leukopoiesis (WBCs)
       • Thrombopoiesis (thrombocytes, aka platelets)
     • Begins during the 20th week of life in the foetal liver and spleen - this role is then taken over by the bone marrow in the young on into adulthood

2. The bone marrow

   • i.e. the site of haemopoiesis (in adults) - restricted to the proximal regions of long bones and the support skeleton
   • Develops in the embryo - from hollowing out of the long bones (forming a central cavity)
     • In the cavity a primitive, undifferntiated cell (haemocytoblast), or stem cell develops
       • All other blood cells are derived from this precursor (multipotent stem cell)
         • Totipotent (omnipotent) = embryonic blastocyst cells that can form ALL cells of the body and cells of the placental (not body) required for viability
         • Pluripotent = can form ALL cells of the body only
         • Multipotent = limited to produce ALL cells of a particular lineage (e.g. blood cells)
   • A combination of two tissues
     • The highly vascular red marrow (50%) is the site of blood cell production
     • The fatty yellow marrow (50%) - no blood production function
     • The 50:50 ratio can change and is measured as the level of marrow activity (marrow cellularity)
   • Blood cell development in the marrow
     • Immature cells are the largest and posses nucleoli (artefacts seen within the nucleus - mainly RNA in structure and are involved in mitosis)
       • In granulated cells (WBCs - neutrophils, basophils, eosinophils) the granules are acquired later in maturation
       • Erythrocyte nuclei are lost later in development
       • Developing cells are retained within the marrow until they are developed sufficiently and functionally capable
         • They are then released into the circulating bloodfor the remainder of their lifespan

3. Mechanism of haemopoiesis

   • Stem cells
     • All blood cells are produced by the bone marrow
       • They all come from a multipotent stem cell (haematopoietic stem cell, HSC)
         • Give rise to...
           • Myeloid cell lineage (RBCs, thrombocytes, granulocytes)
           • Lymphoid cell lineage (lymphocytes - B and T)
         • Self renewal / commitment into a commited cell lineage progenitor
           • Commitment -> progenitor cell 1 (e.g. common lyphoid progenitor)
             • Colony forming unit (CFU)
               • Division and differentiation
                 • Morphologically distinct intermediate blast cell
                   • Maturation
                     • Mature blood cell type B
                 • Morphologically distinct intermediate blast cell
                   • Maturation
                     • Mature blood cell type A
           • Renewed HSC (stem cell renewal)
             • ->
           • Commitment -> progenitor cell 2 (e.g. comon myeloid progenitor)
             • Production of lineage blood cells
       • The bone marrow and spleen form a supportive environment (called the haemopoietic microenvironment)
     • Pluripotent stem cell (from developing blastocyst)
       • Multipotent stem cell (HSC in bone marrow)
         • Diameter 18-23um (large)
         • Give rise to myeloid and lymphoid progenitors
           • (Common) Myeloid progenitor
             • RBCs, granulocytes (PMNs, eosinophils, basophils) and thrombocytes
           • (Common) lymphoid progenitor
             • Lymphocytes only
           • Progenitor cells
             • = irreversibly commited stem cell (cannot self-renew)
               • Regulated by certain hormones or substances so that they can proliferate and undergo maturation
   • Growth factors (GFs)
     • Haemopoiesis occurs in a suitable microenvironment provided by a stromal matrix on which stem cells grow and divide
       • There are probably specific recognition and adhesion sites sites
         • Extracellular glycoproteins and other compounds are involved in the binding
       • Microenvironment (adhesion and growth factors) provided by local macrophages, ECM, adipocytes, fibroblasts etc.
     • E.g. granulocyte colony stimulating factor (G-CSF) - in the development and maturation of neutrophils (from myeloid progenitor)
       • Stimulates proliferation of early bone marrow cells
         • Causes direct differentiation to one or other cell type
           • Stimulates cell maturation (in neutrophil single nucleus beomces lobed; normal = 3-5 lobes)
             • Supress apoptosis or affect the function of mature non-dividing cells
       • Binds to cells expressing the correct receptor
         • Signalling via ...
           • JAK/STAT
           • JAK/Ras -> myc/fos pathway
           • JAK/PI3K -> Akt (blocks apoptosis)
   • Regulators
     • Important in the differentiation of colony forming unit lineages
       • Burst promoting activity
         • (Erythroid) BFU-E cell line
       • Erythropoietin
         • (Erythroid) CFU-E cell line
         • A glycoprotein hormone (produced in the kidneys) - stimulates the production of RBCs by stem cells i bone marrow
           • Secretion is stimulated in response to low O2 (by the juxtaglomerular O2 sensor)
             • Secreted to balance RBC production with RBC loss
       • Colony stimulating factor (CSF)
         • (Granulocyte-monocyte) CFU-GM cell line
       • Megakaryocytic stimulating activity
         • (Megakaryocytes) CFU-M cell line
       • Thrombopoietin
         • Megakaryocytes
         • (TPO) - the major cytokine (glycoprotein hormone) that determines the proliferation of megakaryocytes (produce platelets)
           • Produced in liver and kidneys

4. Blood cell development

   • Primitive stem cell (haemacytoblast)
     • Erythrocyte series
       • Erythrocyte development
         • HSC
           • Common myeloid progenitor
             • Burst promoting activity
               • +
             • BFU-E
               • CFU-E
                 • Rubriblast (proerythroblast)
                   • First identifiable RBC precursor
                   • Prorubricyte
                     • Rubricyte (normoblast)
                       • Looks like a monocyte - but much smaller
                       • Metarubricyte (loses nucleus at this stage)
                         • Reticulocyte (aka diffusely basophilic erythrocyte)
                           • Erythrocyte (mature red cell)
               • Erythropoietin (EPO)
                 • +
     • Leukocyte series
     • Thrombocyte series

5. Different blood cell types

   • Erythrocytes
     • Biconcave disc
     • Approximately 7um in diameter
     • Non-nucleated
     • Contains haemoglobin (Hb)
     • Major function is O2/CO2 transport
   • Leukocytes
     • Granulocytes (Myeloid)
       • Neutrophils
         • 12-15um diameter
         • characterised by segemented nucleus (2-5 lobes = normal)
         • Dense chromatin with regions of parachromatin (lighter areas) = lobes and nodes
         • Slightly eosinophilic (pinky) cytoplasm
         • Important in inflammatory processes (phagocytes and mediators)
       • Eosinophils
         • Most readily reconisable - due to orange granules
         • 12-15um diameter
         • 2-3 lobes to nucleus
         • Normally represent 0-6% of WBCs in blood
         • Granules contain; major basic protein (MBP) - toxic to parasites
       • Basophil
         • Contain purple/black large granules (may obscure the nucelus)
         • 2-3 lobes to nucleus
         • 12-15um diameter
         • Contain; heparin and large amaounts of histamine (involved in hypersensitivity reactions - mediated inflammation to antigen responses)
       • Monocyes
         • Largest cells in peripheral blood (12-20um)
         • Abundant blue/grey (sometimes pinky) cytoplasm, with small granules
         • Fine reddist granules may be seen
         • Phagocytes - frequently contain vacuoles
         • Can migrate into tissues (where they are called macrophages)
         • Central role in the inflammatroy process - replace neutrophils during chronic inflammation
     • Lymphocytes (Lymphoid)
       • Vary in size (7-20um) and nuclear:cytoplasmic ratio
         • Small lymphocytes (7-12um) have rounded nucleus with dense chromatin and little cytoplasm
         • Can appear much larger (15-20um) with more cytoplasm and irregularly-round nucleus
       • Represent 15-60% of peripheral blood

6. Bone marrow sampling and analysis

   • Myeloid:erythroid (ME) cell ratios
     • Provides information on the relative numbers of WBCs, RBCs and their precursors
     • In a healthy adult the number of WBC precursors exceeds the number of RBC precursors by 3 or 4 to 1
       • This ratio changes in disease (increased in infection, dominated by myelocytes and other WBC precursors in leukaemia)
   • Cytochemistry, flow cytometry, electronic resistance and lightscattering
     • Cytochemistry - chemical stains that react with cytoplasmic components and define different cells in blood and bone marrow
       • Blood cells contain multiple enzymes, fats and other substances that can be detected (important in the diagnosis of myelocytic leukaemias)
         • FAB( french-american-british) classification of acute leukaemias is based on morphology and cytochemistry
       • Most important cytochemical studies in the study of acute leukaemia are
         • Myeloperoxidase (MPO)
           • Lysosomal enzyme in myelocytes
             • Staining can reveal acute leukaemias (of myeloid lineage)
         • Non-specific esterase
         • Acid phosphatase (AP)
           • Stains lysosomal acid phosphatase - can detect hair cell leukaemia
         • Periodic acid schiff (PAS) stain
           • Stains polysaccharides (e.g. glycogen)
     • Flow cytometry - allows identification of blood cells according to different molecules on the cells
     • Electronic resistance
       • Cells pass through aperture with electric current across it (changes in current due to differernt cell sizes detected) - automated counting
     • Light scattering
       • Cells counted as they pass through a focused laser
         • Results sum of
           • Diffraction - bending of light around corners
           • Refraction - bending due to change in speed
           • Reflection - light rays reflected back by obstructive structures