BLOOD AND HEMATOPOIESIS https://static.wixstatic.com/media/f7d5d5_556b193d57884373904c39dff4f187d6~mv2.jpg/v1/fill/w_646,h_363,al_c,q_80,usm_0.66_1.00_0.01/f7d5d5_556b193d57884373904c39dff4f187d6~mv2.webp Department of Histology and Embryology LF MU Petr Vaňhara BLOOD Blood is body fluid • transport medium (O2, CO2, metabolites, hormones, nutrients...) • homeostasis of inner body environment (thermoregulation, acidobasic equilibrium, oncotic pressure) • integrity of cardiovascular system (clotting cascade) • immune reactions formed blood elements • erythrocytes • leukocytes • thrombocytes plasma • ions, proteins, low mass organic compounds • fluid ECM Blood can be considered as a specialized connective tissue BLOOD PLASMA AND TISSUE FLUID plasma • 2,8-3,5 l • pH 7.4 ( 0.05) •  92% water •  1% ions (Na+, K+, Ca+, Mg+, Cl-, HCO3 -), low mass organic compounds (glucose, aminoacids, cholesterol, lipids, waste products of metabolism), respiration gases •  7% proteins (albumins, globulins, fibrinogen) Venous blood Lung capillaries Arterial blood Capillaries Venous blood IONS AND LOW MASS MOLECULES OF BLOOD PLSAMA (1%) •  1% ions (Na+, K+, Ca+, Mg+, Cl-, HCO3 -), low mass organic compounds (glucose, aminoacids, cholesterol, lipids, waste products of metabolism), respiration gases Cations Sodium 136–148 mmol/l Osmotic pressure, volume, pH Potassium 3,7–5,0 mmol/l Membrane potential of cells (nerve, muscle) Calcium 2,15–2,61 mmol/l Permeability of membranes, blood clotting, neuromuscular junctions Magnesium 0,66–0,94 mmol/l Cofactor of enzymes, neuronal conduction Iron ♂ Iron ♀ 12–27 μmol/l 10–24 μmol/l Cofactor of enzymes, in hem of hemoglobin Copper 12–22 μmol/l Cofactor of enzymes Anions Chlorides 95–110 mmol/l Osmotic pressure, volume, pH Bicarbonates [HCO3]- 22–26 mmol/l Transport of CO2, buffer - pH Pi 0,6–1,4 mmol/l Buffer - pH Iodide 276–630 μmol/l Hormones of thyroid gland Memorizing of this table is not necesasry for completing our course ☺ IONS AND LOW MASS MOLECULES OF BLOOD PLSAMA (1%) Glucose 3,3–6,1 mmol/l Aminoacids 2,3–3,9 mmol/l Urea 3,0–7,6 mmol/l Lipids 4–9 g/l Triacylglycerols 0,5–1,8 mmol/l Phospholipids 1,8–2,5 g/l Creatinine 55–110 μmol/l Cholesterol (total) 3,5–5,2 mmol/l Bilirubin 3,3–18,0 μmol/l Lactate 0,55–2,22 mmol/l COMPOSITION OF BLOOD PLASMA IS CONSTANT and it is regulated in narrow range → essential for clinical medicine •  1% ions (Na+, K+, Ca+, Mg+, Cl-, HCO3 -), low mass organic compounds (glucose, aminoacids, cholesterol, lipids, waste products of metabolism), respiration gases But what is important to know is that: PROTEINS OF BLOOD PLASMA (7%) • oncotic blood pressure • transport • coagulation • immune response • regulatory proteins https://pharmaceuticalintelligence.files.wordpress.com/2014/06/major_plasma_proteins2.jpg PROTEINS OF BLOOD PLASMA (7%) • prealbumin - transport • albumin - 68kDa - transport - osmotic pressure • 1 region - 1 lipoprotein (HDL) - 1 acid glycoprotein - 1 antitrypsin - (1 fetoprotein) • 2 region - 2 macroglobulin - haptoglobin • 1 region - transferrin - hemopexin -  lipoprotein (LDL) - C4 (complement) • 2 region - CRP - fibrinogen - 2 microglobuin - C3 (complement) •  region - IgA, IgG, IgM  ̶ Why it is useful? Click here. https://en.wikipedia.org/wiki/Reference_ranges_for_blood_tests https://en.wikipedia.org/wiki/Reference_ranges_for_blood_tests https://en.wikipedia.org/wiki/Reference_ranges_for_blood_tests https://en.wikipedia.org/wiki/Reference_ranges_for_blood_tests • serum ≠ plasma BLOOD PLASMA AND SERUM FORMED BLOOD ELEMENTS Leukocytes 4.5-11  103/l neutrophils 71% eosinophils 3% bazophils 1% monocytes 5% lymfocytes 20% Thrombocytes 150-450 103/l Erythrocytes 4.2-6.2  106/l 55% 44% 1% Plasma Erythrocytes HEMATOCRIT Erythrocytes 4.2-6.2  106/l 475% HEMATOCRIT 424% Ratio of erythrocyte mass volume to volume of full blood ♂ ♀ Plasma Buffy coat Erythrocytes Normal Erythropenia Polycythemia ERYTHROCYTES Size depends on osmotic pressure of environment 7,5 2,6 0,75 Deviations from normal size ERYTHROCYTES • anisocytosis - macrocytes (>9 µm) - microcytes (<6 µm) ERYTHROCYTES Erythrocyte is amazingly flexible cell ERYTHROCYTES Shape of erythrocytes • integral proteins - band 3, glycoprotein A (ion transporters) • spectrin • ankyrin • actin and actin associated proteins - tropomodulin, tropomyosin • hemoglobin Deviations from biconcave shape • poikilocytosis - acantocytes (irregular spikes) - codocytes („tyre “) - echinocytes (spiked membrane) - eliptocytes (elliptic) - spherocytes (spheroidal) - stomatocytes (some parts missing or other irregularities) - drepanocytes (sickle) - dacrocytes (tear drop) ERYTHROCYTES Deviations from biconcave shape ERYTHROCYTES Normal Spherocyte Echinocyte Codocyte ERYTHROCYTES Hereditary eliptocytosis ERYTHROCYTES Hereditary eliptocytosis Hereditary spherocytosis ERYTHROCYTES Sickle cell anemia • Abnormal hemoglobin (hemoglobin S) ERYTHROCYTES Sickle cell anemia Malaria • pathological genotype (heterozygote HbS/HbA) is beneficial ERYTHROCYTES • Erythrocytes lack nucleus and organelles • Anaerobic glykolysis • Hemoglobin − Hem (haem, porphyrin) − 4 globular subunits − iron: cycle between Fe2+/Fe3+ − Bohr effect − oxyhemoglobin, deoxyhemoglobin − methemoglobin, carboxyhemoglobin ERYTHROCYTES ERYTHROCYTES • Life span 120 days • Constant abrasion • No regeneration • Removal of aged or damaged erythrocytes in bone marrow and spleen LEUKOCYTES • immune response • morphological classification – cytoplasmic granules (does not follow hematopoesis) Granulocytes Agranulocytes Neutrophils Eosinophils Basophils Monocytes Lymphocytes GRANULOCYTES • Primary (azurophilic) granules derived from lysosomes (= nonspecific granules) • Specific (secondary) granules • Polymorphic nucleus • Terminally differentiated • Short lifespan (hours) • Reduced ER, GA, mitochondria (anaerobic glycolysis) • Apoptosis NeutrophilEosinophilBasophil NEUTROPHILIC GRANULOCYTES • Neutrophils - 50-70% of leukocytes in circulation -  >12 m - Segmented nucleus - Barr’s body in females - Azurophilic (primary) granules - myeloperoxidase, lysozyme, proteases, defensins - Neutrophilic (secondary) granules - collagenase, bactericidal enzymes - Chemotaxis of other leukocytes - Microphages • Neutrophilic band • Neutrophilic segment NEUTROPHILIC GRANULOCYTES NEUTROPHILIC GRANULOCYTES Hunters NETs (neutrophil extracellulat traps) Special form of cell death – „netosis“ NEUTROPHILIC GRANULOCYTES • Extravasation (diapedesis) EOSINOPHILIC GRANULOCYTES • Eosinophils - 1-4% of leukocytes in circulation -  12-15 m - Irregular, characteristic bi-segmented nucleus - Azurophilic (primary) granules - myeloperoxidase, lysozyme, proteases, defensins - Eosinophilic (secondary) granules - bright red (eosinophilic) - major acidic protein - peroxidase - cytokines, chemokines - Chemotaxis of other leukocytes - Phagocytosis of antibody-antigen complexes - Parasitic infections, allergic reaction - Chronic inflammation BASOPHILIC GRANULOCYTES • Basophils - <1% of leukocytes in circulation -  12 m - Irregular, bisegmented nucleus, masked by granules - Azurophilic (primary) granules - myeloperoxidase, lysozyme, proteases, defensins - Basophilic (secondary) granules - 0.5 m - large, dark (basophilic) - heparin, histamin - vasodilatation - phospholipase A - Analogs of mast cells - Receptors for IgE - Allergy, anaphylaxis, inflammation AGRANULOCYTES • Only non-specific, azurophilic granules present • Specific granules absent • Non-segmented nucleus MonocytesLymfocytes LYMPHOCYTES • Round dominant nucleus • Basophilic cytoplasm • Variable lifespan • Biologically different lymphocytes (B, T, NK) are morphologically indistinguishable • Small, medium, large • Bone marrow, thymus • BCR/TCR/Antibodies with amazing variability LYMPHOCYTES • Plasma cells MONOCYTES •  12-15 m • Circulating precursors of macrophages, osteoclasts, microglia, Kupfer cells and dendritic cells • Mononuclear phagocytic system • Large, oval (bean, kidney) nucleus with less condensed chromatin and 2-3 nucleoli • Basophilic cytoplasm • Azurophilic granules SECCHI-LECAQUE / ROUSSEL-UCLAF / CNRI / SCIENCE PHOTO LIBRARYDENNIS KUNKEL MICROSCOPY / SCIENCE PHOTO LIBRARY MACROPHAGES •  around 21 m • variable migratory morphology • phagocytocys • antigen presentation to T-lymphocytes • inflammatary response • tissue regeneration and wound healing • e.g. histiocytes, Kupffer cells, microglia DENDRITIC CELLS • „professional“ presentation of antigens to immune cells (MHC II) • activation or inhibition of lymphocytes • immune response or immunetollerance • e.g. DC in epidermis (Langerhans cells) THROMBOCYTES • Cell fragments without nucleus •  2-3 m, discoid shape • hyalomere, granulomere • 150-400  103/l • blood clotting, repair of vessel wall -granules 300-500 nm fibrinogen, PDGF -granules 250-300 nm serotonin, CaII+ pyrophosphate ADP, ATP -granules 175-200 nm lyzosomal enzymes THROMBOCYTES 1. Primary aggregation of platelets - collagen fibers exposed by endothelial rupture - platelet clot 2. Secondary aggregation of platelets - clotting factors, ADP from thrombocytes attracts other platelets – white thrombus 3. Coagulation – blood clotting - fibrin mesh capturing erythrocytes – red thrombus 4. Thrombus retraction - contraction of thrombus (platelet actin and myosin) 5. Thrombolysis - dissolving of thrombus (plasmin) and tissue regeneration THROMBOCYTES You needn’t to know the clotting cascade in full details for our course, leave it for biochemistry ☺ Neutrophil band 4 % segment 67 % 1:17 shift to the left shift to the right more bands more segments Eosinophils 3 % Basophils 1 % Lymphocytes 20 % Monocytes 5 % ∑ = 100 % DIFFERENTIAL WHITE BLOOD CELL COUNT Norm THIS SLIDE IS REALLY IMPORTANT  Increased  Decreased Neutrophils neutrophil granulocytosis neutrophil granulocytopenia Eosinophils eosinophil granulocytosis eosinophil granulocytopenia Basophils basophil granulocytosis basophil granulocytopenia Lymphocytes lymphocytosis lymphocytopenia Monocytes monocytosis monocytopenia DIFFERENTIAL WHITE BLOOD CELL COUNT Deviations from norm DIFFERENTIAL WHITE BLOOD CELL COUNT Neutrophils bands 0-5 % segments 35-85 % Eosinopils 0-4 % Basophils 0-1 % Lymphocytes 20-50 % Monocytes 2-6 % According to: Haferlach et al. Kapesní atlas hematologie. Grada 2014 Example of population variability DIFFERENTIAL WHITE BLOOD CELL COUNT Wikipedia DIFFERENTIAL WHITE BLOOD CELL COUNT Age dependence https://obgynkey.com/elevated-or-depressed-white-blood-cell-count/ HEMATOPOIESIS ERYTHROPOIESIS • 2  1011 of new erythroyctes daily • proerythroblast (14-19 µm) - mitotically active - dominant, round nucleus with 1-2 nucleoli - mildly basophilic cytoplasm • basophilic erythroblast (13-16 µm) - mitotically active - heterochromatic nucleus with inconspicuous nucleoli - basophilic cytoplasm (sometimes more than in proerythroblast) • polychromatophilic erythroblast (13-16 µm) - mitotically active - production of hemoglobin - blue-gray cytoplasm due to combined basophilic (polyribosomes) and acidophilic aspects (hemoglobin) - heterochromatic nucleus (checkerboard appearance) • orthochromatophilic erythroblast (8-10 µm) - mitotically inactive - small, compact, eccentric, pycnotic nucleus → extrusion - mildly acidophilic cytoplasm with basophilic residues • reticulocyte (polychromatophilic erythrocyte,  7-8 µm) - lacks nucleus, still spheroid shape - acidophilic cytoplasm - substantia reticulofilamentosa visible by supravital staining (brilliant cresyl blue) • erythrocyte (7-8 µm) - anucleate, biconcave disc - acidophilic cytoplasm ERYTHROPOIESIS proerythroblast orthochromatophilic erythroblast erythrocytes orthochromatophilic erythroblast basophilic erythroblast polychromatophilic erythroblastreticulocyte eryhtrocytes GRANULOPOIESIS • myeloblast (15 µm) - mitotically active - round-oval, euchromatic nucleus - 2-6 apparent nucleoli - weakly basophilic cytoplasm without granules • promyelocyte (15-24 µm) - mitotically active - round-oval nucleus with partly condensed chromatin - basophilic cytoplasm with azurophilic granules • neutrophilic, eosinophilic or basophilic myelocyte (10-16 µm) - mitotically active - oval or bean-shaped nucleus with condensed chromatin - increasing number of specific granules in cytoplasm • neutrophilic, eosinophilic or basophilic metamyleocyte (10-12 µm) - mitotically inactive - horseshoe-like nucleus with condensed chromatin • neutrophilic, eosinophilic or basophilic granulocyte (10-12 µm) - segmentation of nucleus - cytoplasm rich in specific and azurophilic granules myeloblast promyelocyte myelocyte metamyelocyte granulocyte-band granulocyte-segment myeloblast promyelocyte promyelocyte neutrophilic myelocyte promyelocyte eosinophilic myelocyte neutrophilic myelocyte neutrophilic metamyelocyte neutrophilic segment polychromatophilic erythoblast GRANULOPOIESIS myeloblast promyelocyte myelocyte metamyelocyte granulocyte THROMBOPOIESIS • megakaryoblast (up to 30 µm) - large oval, nonlobed nucleus with prominent nucleoli - basophilic cytoplasm - successive endomitoses without karyokinesis and cytokinesis • promegakaryocyte (up to 100 µm) - large cell with polyploid nucleus (8n-64n) • megakaryocyte (80-150 µm) - polyploid, multilobed nucleus (8n-64n) - azurophilic and platelet granules - multiple centrioles, ER and Golgi apparatus - numerous peripheral invaginations of plasma membrane ̶ platelet demarcation channels defining individual thrombocytes - release of thrombocytes into bone marrow sinusoids MONOCYTOPOIESIS • monoblast (16 µm) - round, bean shaped nucleus with 2-6 nucleoli - mildly basophilic cytoplasm • promonocyte (16-20 µm) - mitotically active (1-2 divisions) - large nucleus with mild indentation, unapparent nucleoli - basophilic cytoplasm - azurophilic granules • monocyte - short-time in circulation, then extravasation and differentiation to tissue macrophages LYMPHOPOIESIS • lymphoblast (18-20 µm) - round-oval nucleus with several nucleoli - mildly-basophilic cytoplasm without azurophilic granules • prolymphocyte (12-15 µm) - morphological transition and maturation to lymphocytes • lymphocyte - further maturation and differentiation outside bone marrow MONOCYTOPOIESIS AND LYMPHOPOIESIS OVERVIEW OF ADULT HEMATOPOIESIS Osteoclasts CD34+ HEMATOPOIETIC STEM CELLS AND PROGENITORS • Hematopoietic stem cell - Quiescent, slow cell cyle - Bone marrow niche - Transmembrane phosphoglycoprotein CD34+ - adhesion within niche - No expression of lineage surface markers (Lineage negative or Lin-) - Autologous transplantations • Colony/Burst – Forming Unit – CFU/BFU - Progenitors of individual lines characterized in vitro - Collonies in vitro EMBRYONIC HEMATOPOIESIS • Extraembryonic mesoblastic period (day 16-20 – week 8) - yolk sac - classical model – hemangioblasts (bipotent cells) - large, nucleated erythroid cells • aorta-gonad-mesonephros (day 28 – week 4) • hepatolienal period (month 1 – birth) - colonization of fetal liver and spleen • medullary period (month 4-6. – rest of life) - bone marrow • hepatolienal and bone marrow hematopoiesis • erythroblast islands HEMATOPOIETIC ISLANDS • Aorta-gonad-mesonephros (day 28 – week 4) - para-aortic clusters in mesoderm of splanchnopleura - source of embryonic HSCs INTRAEMBRYONIC HEMATOPOIESIS • Placenta SUMMARY OF HEMATOPOIESIS Embryonic - yolk sac - AGM - liver and spleen - bone marrow Adult - bone marrow (yellow, red) - extramedullar hematopoiesis rare (pathology) http://dev.biologists.org/content/133/19/3733 Thank you for attention pvanhara@med.muni.cz www.med.muni.cz/histology