Blood and haemopoiesis Lecture 2010

Blood Volume – approximately 5,5 L Hematocrit – volume of the erythrocyte mass: women 35 – 45%, men 40 – 50% Buffy coat – 1% - leukocytes and thrombocytes Rest – plasma (water, inorganic salts, organic compounds, proteins (albumin, alfa, beta, gama globulins, lipoproteins, fibrinogen) Serum – without coagulum (fibrinogen and factors for haemostasis).

Erythrocytes Biconcave discs without nuclei Size: 7,5 x 2,6 μ m (in the middle only 0,8 μ m) Shape guarantees larger surface that is important for better gas exchange Number: 3,9 – 5,5 milions in 1 μ L It depends on age, gender, race and othe factors as diet,altitude ect. Therefore, every country has to determinate and statistically calculate reference values. Plasmalemma, cytoskeleton, hemoglobin (33%), enzymes: glycolysis, hexose- monophosphate shunt They survive 120 days in circulation Oligosacharides on the surface – AB0 system

Normal hematocrit Children: 1-7 days 0,420-0, weeks 0,390-0, weeks 0,360-0, months 0,300-0,480 0,5-2 years 0,330-0, years 0,330-0, years 0,350-0, years 0,360-0,490 Men: years 0,370-0,490 Women: years 0,360-0,460 Men: years 0,390-0,510 Women: years 0,330-0,470

Erythrocytes in 10 * 12/l 1-7 days 3,9-5, weeks 3,60-6, weeks 3,20-5, months 2,90-4,90 0,5-2 years 3,70-5, years 3,90-5, years 4,0-5, years 4,10-5,30 Men: years 4,19-5,75 Women: years 3,54-5,18 Men: years 4,19-5,75 Women: years 3,54-5,18

Medical application Anemia Polycythemia – physiological adaptation or haemoblastosis – it increases blood viscosity Anisocytosis – higher variability in size Macrocytes – greater – impairment of maturation (deficiency of vit. B12) Microcytes – smaller (defeiciency of iron) Sicle cell disease – point mutation in the gene for hemoglobin

Leukocytes in 1 μL Differencial count: Granulocytes Neutrophiles % Eosinophiles 2-4% Basophiles 0-1% Agranulocytes Lymphocytes 20-30% Monocytes 3-8%

Leukocytes (10*9/l ) 1-2 days 9,00-38, days 5,00-21, weeks 5,00-19, months 5,50-19,00 0,5-2 years 6,00-17, years 5,00-17, years 4,50-14, years 4,50-13, years 4,50-11,00 Men: years 4,10-10,20 Women: years 4,00-10,70

Granulocytes Content of two granule types: Specific with specific function for given leukocyte type azurophilic (lysosomes) All granulocytes in periferal blood are terminal postmitotic stages of the cell development – cells do not divide. They survive only few days in peripheral blood Nuclei are divided in two or more lobes

Neutrophilic granulocytes (polymorphonuclear) 60-70% Size: μ m (in smear) Nucleus is divides in two or three lobes that are connected by chromatin briges; inactive X chromosome – Barr body Specific granules – small, near the limit of resolution in SM - lysosomes Azurophilic granules – 0,5 μ m Glycogen – source of energy – anaerobic metabolism Phagocytosis, oxidative burst – (H 2 O 2 ) and oxygen radicals

Eosinophilic granulocytes 2-4% Size: μ m Bilobed nucleus Specific granules: crystalline core (internum) – major basic protein matrix (externum) - lighter Alergy, parasitic infections Modulation of inflamation (inactivation of leukotrienes and histamine)

Basophilic granulocytes Less that 1% Size μ m Nucleus is divided in irregular lobes, but it is not distinct because it is covered by granules Specific granules – metachromatic, content of heparine and histamine – liberation of granules – degranulation - after binding of certain antigens.

Agranulocytes They do not contain specific granules, only azurophilic Nucleus is round or indented – kidney shaped Lymphocytes Monocytes

Lymphocytes Division according to the size: small (6-8 μ m), medium-sized and large (up to 18 μ m). Small lymphocytes prevail in blood – memory cells Large round nucleus, chromatin is condensed, nucleolus Thin rim of cytoplasm, ribosomes, azurophilic granules

Function T and B lymphocytes – specific immunity (T lymphocytes prevail in peripheral blood: 65% - 75% ) B lymphocytes – humoral – differentiation in plasma cells – production of antibodies T lymphocytes – cytotoxic CD8 (induce apoptosis), helper CD4 (cooperate with B-lymphocytes and macrophages) NK (Natural killers) lymphocytes – 10-15% in peripheral blood – nonspecific - innate immune response

Monocytes Size: μ m Oval, horseshoe or kidney shaped nucleus, excentrically placed Basophilic cytoplasm, azurophilic granules (lysosomes), RER, polyribosomes, mitochondria, Golgi complex Monocytes differentiate into macrophages (antigen presenting cells)

Platelets - thrombocytes Nonnucleated, disc-like cell fragments of megakaryocytes Size μ m – in uL Central zone containing granules – granulomera and peripheral lighter zone - hyalomera Hyalomera: open canalicular system – invagination of superficial plasmalemma, marginal bundle of microtubules, actin and myosin – contractile Granulomera: mitochondria, glycogen, granules ( δ - dense bodies – ATP and serotonin; α – platelet-specific proteins: fibrinogen, PdGF, FGF, von Willebrant factor, platelet- specific factor IV; λ - lysosomes)

Primary aggregation – platelet plug Secondary aggregation – release of granules Blood coagulation – activation of cascade – gives arise to fibrin – thrombus = blood clot Clot retraction (actin and myosin) Clot removal – plasmin Medical application = hemophilia – inherited deficiency of factors Function – hemostasis:

Hematopoiesis 1. stage – development of cells in mesoderm of yolk sac – blood islets 2. stage - hepato-lienal period 3. stage – bone marrow Hematopoietic stem cells – pluripotent stem cells - self-renewing – low mitotic activity Unipotent progenitor cells - self-renewing – high mitotic activity Precursor cells (blasts) – not self-renewing

Hematopoiesis Hematopoiesis is a result of simultaneous proliferatiuon and differenciation Pluripotent stem cell – haemocytoblast Lymphoid and myeloid multipotent stem cells Unipotent stem cells – colony forming cells Precursors - blasts (lymphoblast, erythroblast....) Mature cells – cytes (lymphocyte, erythrocyte)

Development of blood cells depends on: Microenvironment – cells of stroma, extracellular matrix Growth factors: growth factors – stimulation of mitotic activity colony-stimulating factors (CSF) hematopoietins (erythropoietin)

Red bone marrow Stroma: hematopoietic cords and sinusoids Stroma – reticular connective tissue (reticular cells and reticular fibres – (collagen type 1 and 3, fibronectin, laminin and proteoglycans) Sinusoids – capillaries with discontinuous endothelium Stem cells – they can differentiate also in other cellular types that blood cells – appropriate stimulation is necessary

Erythropoiesis Erythropoietin, iron, folic acid, cyancobalamin (vit B12) division from proerythroblast to erythrocyte Approximately 7 days from proerythroblast to reticulocyte Decrease of the cell volume Condensation of chromatin (pycnotic nucleus) and its expulsion Synthesis of hemoglobin and successive loss of basophilia (polyribosomes)

Erythropoiesis Proerythroblast – large cell with loose chromatin, nucleoli and basophilic cytoplasm Basophilic erythroblast – condensed nucleus, basophilic cytoplasm - polyribosomes Polychromatophilic erythroblast – polyribosomes decrease, hemoglobin appears Orthochromatophilic erythroblast – eosinophilic cytoplasm- hemoglobin, maximally condensed nucleus – nucleus expulsion Reticulocyte – rest of ribosomes – substantia reticulo- filamentosa – 1% in peripheral blood Erythrocyte

Granulopoiesis Occurence of azurophilic and later also specific granules Myeloblast – finely dispersed chromatin, no granules Promyelocyte – basophilic cytoplasm, Golgi complex and azurophilic granules Neutrophilic, basophilic and eosinophilic myelocytes – condensation of nucleus, appearance of specific granules Neutrophilic metamyelocyte – band cell - mature granulocyte

Kinetics Myeloblast – mature neutrophilic leukocyte – 11 days Medullary formation compartment in bone marrow – mitosis and maturation Medullary storage compartment - release on demand Circulating compartment Marginating compartment – adhesion to the endothelium Diapedesis through the endothelium of capillaries and postcapillary venules into the connective tissue (1-4 days - apoptosis)

Maturation of lymphocytes No specific morphologic differences Progenitors in bone marrow – lymphoblasts, prolymphocytes – they do not have antigens specific for B and T lymphocytes Maturation in thymus (T) or in bone marrow (B), they may divide in periphery in lymphoid organs (spleen, lymph node, tonsil)

Maturation of monocytes Monoblast – identical with myeloblast Promonocyte – large cell – up to 18 μ m; basophilic cytoplasm, large slightly indented nucleus, large amount of RER, large Golgi complex Formation of azurophilic granules – lysosomes In blood, they circulate approximately 8 hours They survive in periphery for several months – macrophages - antigen presenting cells

Origin of platelets Megakaryoblast – μ m, large ovoid nucleus, numerous nucleoli. Polyploid cell - up to 30 sets of chromosomes. Megakaryocyte – giant cell - 35 – 150 μ m. Irregular nucleus, numerous mitochondria, well- developed RER and extensive Golgi complex. Formation of granules. Invagination of plasmalemma – demarcation membranes – they surround areas – processes – fragmentation – release of platelets into the blood After dessintegration of cytoplasm into the platelets, megakaryocyte die by apoptosis