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Blood 160 SFST Blood ● Blood smear ● ● Differential count Bone marrow smear Blood ● ● ● ● ● Volume – approximately 5,5 L Hematocrit – volume of erythrocytes (percentage): 35 – 45% in women, 40 – 50% in men 1% leucocytes and platelets (buffy coat) Rest – plasma (water, inorganic salts, organic compounds, proteins (albumin, alfa, beta, gama globulins, lipoproteins, fibrinogen) Serum – after removal of fibrinogen and coagulation factors Blood smear ● ● ● ● Droplet of peripheral blood on the slide Dry, then stain according to Pappenheim: 3 minutes by May-Grünwald solution (fixation by methanol), solve and stain for 1 minute. Then stain by GiemsaRomanovski solution for 15 - 20 minutes. Wash and dry. Observe under immersion objective. Blood smear ● Staining according to Pappenheim: ● May-Grünwald– – – ● Giemsa-Romanovski – – – – ● Eosin-methylene blue - 1 g Methanol - 100 ml Glycerol - 50 ml Azur- eosin II - 3 g Azur II - 0,8 g Glycerol - 250 g Methanol - 250g Result is similar to the hematoxylin eosin staining Erythrocytes ● Biconcave discs without nuclei ● Size: 7,5 x 2,6 μm (in the middle only 0,8 μm) ● Number: 3,9 – 5,5 milions in 1μL ● Plasmalemma, cytoskeleton, hemoglobin (33%), enzymes: glycolysis, hexosemonophosphate shunt ● They survive 120 days in circulation ● Function: transport of gases (O2, CO2) Neutrophilic granulocyte ● 60-70% ● Size: 12-15 μm (in smear) ● ● Nucleus is divided 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 μm ● Glycogen – source of energy – anaerobic metabolism ● Function: Nonspecific immunity: phagocytosis, oxidative burst – (H2O2) and oxygen radicals Neutrophilic granulocyte Eosinophilic granulocyte ● 2-4% ● Size: 12-15 μm ● Bilobed nucleus ● Specific granules: crystalline core (internum) – major basic protein matrix (externum) - lighter ● ● ● Function: activity again parasitic infections Modulation of inflamation (inactivation of leukotrienes and histamine) Eosinophilia – typical for parasitic infection, alergy Eosinophilic granulocyte Basophilic granulocyte ● Less that 1% ● Size 12-15 μm ● ● ● 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. Function – modulation of immune response Basophilic granulocyte Lymphocyte ● ● ● ● ● Division according to the size: small (6-8μm), medium-sized and large (up to 18μm). Division according to the specific superficial antigens – B, T, NK 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, helper CD4 NK (Natural killers) lymphocytes – 10-15% in peripheral blood – nonspecific - innate immune response (induce apoptosis) Lymphocyte Monocyte ● ● ● ● ● Size: 12 -20μm 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) Function: phagocytosis, antigen-presenting cells Monocyte Platelet ● Nonnucleated, disclike cell fragments of megakaryocytes ● Size - 2-4 μm ● 200 000– 400 000 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 ● Function: coagulation Platelets Leukocytes ● 6 000 -10 000 in 1μL ● Differencial count: ● Granulocytes ● Neutrophiles 60 -70% ● Eosinophiles 2-4% ● Basophiles ● Agranulocytes ● Lymphocytes 20-30% ● Monocytes 0-1% 3-8% Neutrophilic granulocytes ● ● ● 2,7 – mean of nuclear segments in neutrophilic granulocytes Shift to the left – release of younger neutrophilic granulocytes – sign of the inflamation Shift to the right – more segments – failure of neutrophilic granulocyte development (hemopoiesis) – absence of vitamines Red bone marrow ● ● Stroma – reticular connective tissue (reticular cells and fibres – collagen type 1 and 3, fibronectin, laminin, proteoglycans) Sinusoids – capillaries with discontinuous endothelium Red bone marrow ● ● Stroma: reticular connective tissue – reticular cells and extracellular matrix Capillaries with discontinuous endothelium ● Adipocytes ● Stem cells ● Erythroblastic islands ● Myelopoetic cells ● Megakaryocytes ● Mast cells Hemopoiesis ● ● Erythrocyte development: Proteosyntesis – syntesis of hemoglobin ● Loss of nucleus and ribosomes ● Proerythroblast ● ● ● ● ● Formation of azurophilic and specific granules ● Segmentation of nucleus ● Myeloblast ● Promyelocyte ● Myelocyte Basophilic erythroblast Polychromatophilic erythroblast Orthochromatophilic erythroblast Granulocyte development: ● Reticulocyte ● Metamyelocyte ● Erythrocyte ● Mature leukocyte Hemopoiesis Erythroblastic island