Haemopoiesis

Question 1

Where is the site for haemopoiesis?

Answer 1

○During the first two months, the yolk sac is the site for haemopoiesis.
○From months 2-7 it changes to the liver and spleen.
○From months 5-9 it changes to the bone marrow, which remains as the site during childhood and adulthood.

Question 2

Describe the two types of bone marrow.

Answer 2

○Red marrow contains haemopoietic tissue, while yellow marrow is fat.
○During infancy, all the bone marrow is red, and as aging occurs, red marrow is replaced by yellow marrow.

Question 3

What is the difference between intramedullary and extramedullary?

Answer 3

○Intramedullary describes formation and activation of blood cells in bone marrow, while extramedullary describes formation and activation of blood cells outside bone marrow.
○If bone marrow cannot meet demands of body, due to a pathological condition, haemopoiesis may occur at fecal sites (like the liver, spleen or lymph nodes).

Question 4

Describe the haemopoietic stem cells.

Answer 4

○The pluripotent stem cell can independently replicate, proliferate and differentiate into any lineage.
○Long-term HSCs - haemopoietic stem cells that stay in bone marrow for longer than 16 weeks.
○Short-term HSCs - haemopoietic stem cells that only remain in bone marrow for a few weeks.
○HSCs are CD34+, so although they can’t be viewed under a microscope, they can be identified by flow cytometry.

Question 5

Describe the bone marrow stroma.

Answer 5

○Bone marrow contains sinuses lined within endothelial cells that are vascular spaces or pools of blood.
○Sinuses can control release of mature and immature cells into the blood.
○Also contain non-haemopoietic cells that support bone marrow and produce growth factors.

Question 6

Describe the non-haemopoietic cells in the bone marrow.

Answer 6

○Stromal cells secrete collagen, glycosaminoglycans, fibronectin and thrombospondin to form an extracellular matrix.
○Fibroblasts produce scaffolding that supports other cells.
○Macrophages produce growth factors that promote erythrocyte production and are involved in the storage of iron as well as debris removal.
○Adipocytes store energy as fat.
○Mesenchymal stem cells
○Endothelial cells
○Osteoblasts
○Osteoclasts

Question 7

What is stem cell homing?

Answer 7

○Stem cell homing ensures HSCs stay in the bone marrow by attracting any circulating HSCs, until optimum proliferation occurs.

Question 8

What is stem cell mobilisation?

Answer 8

○Stem cell mobilisation involves releasing the HSCs, for example in response to injury.

Question 9

Describe the general characteristics of groowth factors.

Answer 9

○ Glycoproteins that control proliferation and differentiation of haemopoietic progenitor cells, as well as preventing apoptosis and affecting mature cell function..
○ Can act locally by cell-cell contact or circulate in plasma.
○ Most growth factors are synthesised by stromal cells.
○ Erythropoietin is synthesised mainly by kidneys.
○ Thrombopoietin is synthesised mainly by liver.
○ Multiple growth factors may synergise to cause proliferation or differentiation in a particular cell.
○ One growth factor may cause production of another growth factor or its receptor.

Question 10

Describe adhesion molecules.

Answer 10

○ Glycoproteins that attach cells to each other and to extracellular matrix.
○ Are also involved in cell-cell synapse formation.
○ Made up of three domains, including: intracellular, transmembrane and extracellular.
○ Adhesion molecules in bone marrow connect haemopoietic precursors, leucocytes and platelets to extracellular matrix components, to endothelium, to other surfaces, and to each other.

Question 11

Describe the four groups of adhesion molecules.

Answer 11

○ Integrins - connect extracellular environment (collagen, fibronectin, fibrinogen) to intracellular signalling pathways.
○ Immunoglobulin super family.
○ Selectins - involved in immune system and aid leucocytes in trafficking and homing.
○ Cadherins - maintain tissue structure and function.

Question 12

Describe apoptosis.

Answer 12

○ Programmed cell death is a process where cells activate intracellular proteins that cause cell death.
○ Characterised by cell shrinkage, nuclear chromatin condensation, nucleus fragmentation and DNA cleavage.

Question 13

How is apoptosis initiated?

Answer 13

○ Caused by caspases, which are intracellular cysteine proteases.
○Activated by membrane proteins or cytochrome C.
○Membrane proteins - can include Fas or TNF receptor that cause apoptosis by an intracellular death domain that activates caspases, which digest DNA.
○For ex. Activated cytotoxic T cells express Fas ligand which causes apoptosis in target cells.
○Cytochrome C - released from mitochondria and binds to APAF-1, activating caspases.

Question 14

Describe the role of p53 in apoptosis.

Answer 14

○ Protein p53 is involved in sensing DNA damage.
○ It increases BAX to activate apoptosis, which increases release of cytochrome C.
○ Prevents damaged cell from dividing by stopping cell cycle.
○ Apoptotic cells are then ingested by macrophages.
○ MDM2 is a protein that controls p53 levels.

Question 15

Describe the regulation of apoptosis.

Answer 15

○ BCL-2 prevents apoptosis.
○ A cells susceptibility to apoptosis is determined by intracellular ratio of BAX and BCL-2, which influences cytochrome C release.
○ Growth factors increase BCL-2, causing inhibition of cytochrome C, and thus inhibition of apoptosis.
○ DNA damage increases p53, which increases BAX, causing cytochrome C release, and thus apoptosis.
○ In haemopoietic malignancies, p53 and ATM (cause apoptosis after DNA damage) can be inactivated due to mutations in genes encoding them.

Question 16

Describe erythropoiesis.

Answer 16

○ Production of erythrocytes.
○ Each day, ~10^12 new erythrocytes are produced.
○ Process of a stem cell to a pronormoblast occurs in an erythroid niche.
○ Erythroid niche contains ~30 erythroid cells at varying levels of development surrounding a central macrophage.

Question 17

Describe pronormoblast.

Answer 17

○Large cell.
○Has a dark blue cytoplasm and a central nucleus that contains nucleoli and chromatin.
○Divides several times to form smaller normoblasts.

Question 18

Describe normoblast.

Answer 18

○Contain more haemoglobin.
○Cytoplasm loses RNA and apparatus for protein synthesis, causing it to stain paler blue.
○Nuclear chromatin becomes more condensed.
○Only appear in blood if extramedullary erythropoiesis is occurring or if there is a bone marrow disease, otherwise they don’t appear in human peripheral blood.

Question 19

Describe reticulocytes.

Answer 19

○Removal of nucleus forms a reticulocyte.
○Can still synthesise haemoglobin as it has some rRNA .
○Slightly bigger than an erythrocyte.
○Matures after circulating in peripheral blood for 1-2 days, after it loses all RNA.
○Forms an erythrocyte.

Question 20

Describe erythrocytes.

Answer 20

○A biconcave disk that has no nucleus.
○Stains completely pink.
○16 mature erythrocytes form from one pronormoblast.

Question 21

Describe erythropoietin.

Answer 21

○ A glycosylated polypeptide hormone that regulates erythropoiesis.
○ Mostly produced in kidneys, and 10% produced in liver and other places.
○ Production is stimulated by oxygen tension in kidneys, and isn’t stored beforehand.
○ Decreased oxygen increases production of erythropoietin.

Question 22

How does hypoxia cause production of erythropoietin?

Answer 22

○ PHD2 senses oxygen and uses it to hydroxylate HIFa.
○ This causes vHL protein to break down HIFa.
○During hypoxia, oxygen concentration is low, so PHD2 cannot hydroxylate HIFa.
○ This causes HIF-1a stabilisation, allowing it to from a dimer with HIFB.
○ Dimer causes production of erythropoietin.

Also causes:
○ Formation of new vessels.
○ Formation of glycolytic enzyme.
○ Transferrin receptor synthesis.
○ Decreased synthesis of hepcidin causing increased iron absorption.

Question 23

Describe the effect of erythropoietin.

Answer 23

○ Erythropoietin increases progenitor cells committed to erythropoiesis, in order to stimulate erythropoiesis.
○ GATA2 is a transcription factor that causes erythroid differentiation from pluripotent stem cells.
○ Stimulation of erythropoietin receptor activates transcription factors GATA1 and FOG1.
○GATA1 and FOG1 increase erythroid-specific genes expression (ex. Globin, red cell membrane proteins, and haem biosynthetic membrane proteins).
○ Also increase anti-apoptotic genes expression and expression of transferrin receptor 1.
○ Stimulation of erythropoietin receptors on late BFUE and CFU-E causes them to proliferate, differentiate and produce haemoglobin.
○ Causes increased erythroid cells in bone marrow.
○ Increased oxygen to tissues decreases erythropoietin (negative feedback).
○ Plasma erythropoietin levels can be used in diagnosis - high in anaemia and low in severe renal disease/polycythaemia vera.

Question 24

Describe thrombopoiesis.

Answer 24

○ HSC’s commit to megakaryocyte lineage, and progenitors proliferate.
○ Megakaryocytes undergo differentiation and maturation, which involves nuclear endomitosis (nuclear duplication without cell division), synthesis of granules and organelles, and appearance of demarcation membrane system (DMS), which acts as a membrane reservoir for platelet development.
○ DMS is made up of an extensive network of cisternae and tubules, which are continuous with the plasma membrane.
○ Development of microtubules, actin filaments, spectrin, and other proteins aid proplatelet and platelet formation.
○ Platelets bud off from cytoplasm of megakaryocyte.

Question 25

Describe the actions of thrombopoietin.

Answer 25

○ Glycoprotein mainly produced by liver, but also by kidneys and marrow.
○ Only produced when platelets are needed.
○ Thrombopoietin plasma levels are inversely proportional to platelet and megakaryocyte mass.
○ Regulates megakaryocyte and platelet production.
○It causes differentiation and proliferation of HSCs and increases number of megakaryocytes as well as its size.

Question 26

Describe the regulation of platelet production.

Answer 26

○ Human myeloproliferative leukaemia protein (MPL) gene encodes TPO-R.
○ TPO-R is expressed on surface of HSC’s, megakaryoblasts, megakaryocytes and platelets.
○ Binding of desialylated, senile platelets to hepatic asialoglycoprotein receptor (ASGPR) causes expression of TPO mRNA, causing platelet production.

○ Hepatic thrombopoietin expression can also be caused by IL-6.
○ Megakaryocyte development is also regulated by transcription factors (GATA-!, RUNX1) and cytokines (IL-3, IL-6).

Question 27

Describe the difference between phagocytes and lymphocytes.

Answer 27

○Phagocytes are part of the innate immune response, that act quickly after infection.
○Lymphocytes are part of the adaptive immune response, involved in immunological memory.

Question 28

Describe neutrophils.

Answer 28

○ Characterised by a multi-lobed nucleus (two/five lobes), and a pale cytoplasm that contains granules (stain pink-blue/grey-blue).
○ Short-lived - circulate in peripheral blood for 6-10 hours before moving from capillaries into tissues.
○ Involved in phagocytosis of bacteria and inflammation.

Question 29

Describe the granules of neutrophils.

Answer 29

○ Granules are primary if they appear at promyelocyte stage, or secondary if they appear at myelocyte stage and predominate in mature neutrophil.
○ Both types of granules are lysosomal.
○ Primary granule contains myeloperoxidase and other acid hydrolases.
○ Secondary granule contains lactoferrin, lysozyme and other enzymes.

Question 30

Describe eosinophils.

Answer 30

○ Paler cytoplasm that contains orange granules (orange because it takes up acidic eosin dye when stained).
○ Less than three nuclear lobes - bilobed nucleus.
○ Precursors cannot be distinguished from neutrophil precursors, but eosinophil myelocyte can be recognised.
○ Longer blood transit time compared to neutrophils.
○Involved in allergic responses, defence against parasites and fibrin removal during inflammation.

Question 31

Describe basophils.

Answer 31

○ Only sometimes seen in peripheral blood as only a small amount circulate blood.
○ Many dark cytoplasmic granules which stain purple, and contain heparin and histamine.
○ Lobulated nucleus.
○ Involved in hypersensitivity reactions, and release heparin and histamine.

Question 32

Describe granulopoiesis.

Answer 32

○ After being released from the bone marrow, granulocytes circulate blood for 6-10 hours, after which they enter tissues and perform phagocytic function.
○ They die after 4-5 days during defensive action or by senescence.
○Production increased in response to inflammation, caused by stromal cells and T lymphocytes.

Question 33

Describe the growth factors involved in granulopiesis.

Answer 33

○ Many growth factors are involved in granulopoiesis, including: IL-1, IL-3, IL-5, IL-6, IL-11, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and monocyte CSF (M-CSF).
○ Activate proliferation and differentiation.
○ Affect function of mature cells, such as: phagocytosis, superoxide generation, and cytotoxicity.
○ Also cause inhibition of apoptosis.
○ Increased production of growth factors and cytokines causes increased production of granulocytes and monocytes.

Question 34

Describe monocytes.

Answer 34

○ Bigger than other leucocytes and have a large kidney shaped nucleus with clumped chromatin.
○ Cytoplasm stains blue and contains granules and many fine vacuoles, that give a ground-glass appearance.
○ Precursors are monoblasts and promonocytes, which are difficult to distinguish from myeloblasts and monocytes.
○ Circulate blood for 24 hours, after which they enter tissues (lung, liver).
○ Transform to macrophages and have a life span of several months/years.
○ Macrophages self-replicate in tissues, and are involved in phagocytosis of bacteria and release of cytokines.

Question 35

Describe the growth factors involved in monocyte production.

Answer 35

○ GM-CSF and M-CSF are involved in monocyte production and activation.
○ Inflammation activates monocytes/macrophages, causing them to secrete cytokines.
○ Cytokine release attracts more blood monocytes and other leucocytes to tissue.

Question 36

Describe B cells.

Answer 36

○ Mature in bone marrow, after which they circulate in peripheral blood until they undergo antigen recognition on pathogen.
○ Mediate production of antigen specific immunoglobulins against invasive pathogens.
○ IgA, IgG, IgM, IgD, IgE.
○ B cells can mature into either a memory B cell or plasma cell.
○Plasma cells stay in bone marrow, and are characterised by a round nucleus, a ‘clock-face’ chromatin pattern, and strongly basophilic cytoplasm.
○ Plasma cells express intracellular immunoglobulin and synthesise many antibodies.

Question 37

Describe T cell maturation.

Answer 37

○ Developed from cells that migrated to thymus, and while moving from cortex to medulla differentiated into mature T cells.
○ This process involves deletion of self-reactive T cells and selection of T cells that have specificity for host human leucocyte antigen (HLA).
○ T cell maturation involves presenting certain molecules on cell surface and expression of T cell receptor.
○ T cell receptor is highly variable, and recognises foreign particles or antigens.

Question 38

Describe the two types of T cells.

Answer 38

○ Helper T cells help other immune system cells, and also express CD4.
○ Cytotoxic T cells kill virally infected cells as well as tumours, and also express CD8.

Question 39

Describe the function of T cells.

Answer 39

○Antigen presenting cells (APCs) break down antigens and present broken down peptides of antigen to T cells, as T cells cannot directly bind to antigen.
○ Major histocompatibility complexes (MHCs) are molecules on APC that present the antigen. There are two classes of MHCs: MHC class I, which presents to cytotoxic T cells, and MHC class II, which presents to helper T cells.
○ Aid in antibody production, as well as recognising and destroying virus infected cells.

Question 40

Describe natural killer cells.

Answer 40

○ Cytotoxic lymphocytes that don’t have T-cell receptor and are involved in innate immune response instead of adaptive immune system.
○ Large cells that contain cytoplasmic granules, and express surface molecules CD16, CD56, and CD57.
○ NK cells express HLA molecules receptor on their surface. Target cells that express HLA bind to these receptors and inhibit NK cells. ○However, when target cells don’t express HLA molecules, they cannot inhibit NK cells, and NK cells then kill the target cells.

Question 41

Describe the importance of bone marrow analysis.

Answer 41

○ Measures production of blood cells to help diagnose diseases, including: leukaemia; bone marrow disease; spread of cancer; severe anaemia.

○ Marrow analysis can also tell the success of treatment by sampling bone marrow at different stages of disease, such as after chemotherapy.

○ Conditions of bone marrow can also affect: number, mixture and maturity of cells and fibrosis structure.

○ Early measures, such as full blood count (FBC) and reticulocyte count, aid in evaluating cell production in bone marrow.
○ This can then be compared to current cell populations in the blood.

○ Marrow analysis can also be cultured to identify presence of any microbes.

Question 42

Describe how to obtain bone marrow.

Answer 42

○ Samples can be taken from hip bone, breastbone, lower leg bone and backbone.
○ First the area is cleaned using iodine solution or alcohol.
○ Local anaesthetic is then injected.
○ The bone marrow needle is then placed through skin and into bone.

Question 43

Describe bone marrow aspiration.

Answer 43

○ Bone marrow is smeared on a slide and stained for examination.

○ Romanowsky stain - helps to visualise cell populations and cell morphology.

○ Pearls stain - detects presence of iron in biopsy specimens.
○ For ex. Deposits of ferritin storage in bone marrow biopsy sample.
○ Abnormal in cydroblastic anaemia (blood disorders where body has enough iron but cannot use it to make haemoglobin).
○ Ferric iron stains blue while nuclei stain red.

Question 44

Describe the M/E ratio.

Answer 44

○ The M/E ratio (myeloid to erythroid ratio) is important in diagnosing disease.
○ Myeloid leucocyte precursors are higher in number compared to erythrocyte precursors in a ratio of 3:1 in healthy individuals.
○ M/E ratio can change in different diseases.
○ In myeloid leukaemia, M/E ratio is closer to 2 as bone marrow is dominated by leucocyte precursors.
○ M/E ratio is normal in diseases such as aplastic anaemia, as both myeloid and erythroid precursors are decreased.
○ M/E ratio decreases in diseases where myelopoiesis is suppressed (due to chemotherapy or viral infections), or where erythropoiesis is increased (due to severe blood loss, iron deficient anaemia or polycythaemia vera).

Question 45

Describe fluorescence flow cytometry.

Answer 45

○ Identifies blood and bone marrow cells depending on their size and granularity.
○ Uses monoclonal antibodies that are conjugated with fluorochromes to assess presence of CD molecules (glycoproteins) on cell surface or in cytoplasm.

○ Haemopoietic stem cells - CD34.
○ Leucocytes - CD45.
○ Monocytes - CD14.
○ Myeloid cells - CD13, CD33, CD117.
○ B lymphocytes - CD10, CD19, CD20, CD22.
○ T lymphocytes - CD2, CD3, CD7.