Peripheral Blood PDF

Peripheral Blood Scott D. Blystone, Ph.D.  Erythrocytes (red blood cells) Wright-Giemsa Azure B+ DNA, GAGS Methylene Blue+ RNA Eosin- Protein Romanovsky Stains Erythrocytes (red blood cells) Function: Transport of gases in the blood in dissolved form or bound...

Peripheral Blood Scott D. Blystone, Ph.D.  Erythrocytes (red blood cells) Wright-Giemsa Azure B+ DNA, GAGS Methylene Blue+ RNA Eosin- Protein Romanovsky Stains Erythrocytes (red blood cells) Function: Transport of gases in the blood in dissolved form or bound to hemoglobin. pH homeostasis -carbonic anhydrase: CO2 + H2O HCO3- + H+ -band 3 membrane protein: exchanges HCO3- for extracellular Cl- Number: Males: 4-6 x106/microliter 40-50% Hct Females: 4-5.5 x106/microliter 35-45% Hct Can vary up to 10% with altitude. Life Span in Blood: 120 days Senescent RBCs are removed from circulation by macrophages in the spleen, liver, and bone marrow.  Erythrocytes Biconcave disks -maximizes surface area for gas exchange -stabilized by cytoplasmic protein skeleton -cortical actin, spectrin, ankyrin -deformable for small capillaries -form cup shape in smallest vessels -stack into rouleaux in larger vessels. -membranes are 50% protein -ion transporters including band 3 protein to maintain isotonicity.  Erythrocytes 8.0 micron diameter, thickness is 2 microns at periphery, 1 micron in center -very uniform size provides microscopic measuring standard Intensely eosinophilic in Wright-Giemsa staining -lacking nuclei, ribosomes, mitochondria, ER, Golgi -lighter in center due to variation in thickness  Erythrocytes SEM TEM  Erythrocytes Hemoglobin -about 1/3rd of RBC mass -responsible for eosinophilia of RBCs -globular chromoprotein tetramer with 4 chains, +heme group that binds iron. Forms: Hb A1, 95% of adults, 2 alpha and 2 beta chains Hb A2, 5% of adults, 2 alpha and 2 delta chains Hb F, fetal development, 2 alpha and 2 gamma chains Hb S:Sickle Cell Trait, A variant Hb C: A variant Glu-Val substitution in beta chain decreases Hb solubility Decrease in RBC flexibility Leads to anemia and increases in blood viscosity  Leukocytes (white blood cells) Granulocytes: Neutrophils (60-70%) Eosinophils (2-4%) Basophils (0-1%) Agranulocytes: Lymphocytes (20-30%) Monocytes (3-8%) Marginating pool – selectin:selectin binding. Diapedesis integrin adhesion for adhesion, migration  Neutrophils PMN, Polys, Polymorphonuclear Leukocytes Anti-bacterial Phagocytosis (+ lysosome fusion) Hydrolize bacterial cell wall w/lysozyme Lactoferrin iron binding Netosis Secretion of fibrous structure to restrain bacteria Oxygen radical release Cytokine release TNF-alpha, IL, TGF=beta Recruit other WBC, fibroblasts, smc  Neutrophils 6000/microliter 1 week in blood 15-20 microns diam. Lobed nucleus Condensed chromatin Cytoplasm pink overall Cytoplasm grainy Multiple purple granules  Neutrophils Specific granules (eosinophilic, small) Type IV collagenase (aids migration) Lactoferrin (sequesters iron) Lysozyme (digests bacterial cell wall) Non-specific granules (lysosomes, azurophilic, large) Myeloperoxidase (antimicrobial) Acid hydrolase (>50 enzymes, low pH)  Eosinophils Anti-parasitic Phagocytosis Ab-dependent recognition Major Basic Protein release, larva Pro-inflammatory release leukotrienes, IL Anti-inflammatory inactivate histamine  Eosinophils 250/microliter 1-2 weeks in blood 12-20 micron diam. Bi-lobed nucleus normal Condensed chromatin Clear cytoplasm Very large pink-orange granules  Eosinophils TEM of eosinophils reveals oval specific granules containing a dark crystalloid body composed of Major Basic Protein.  Eosinophils Specific granules (eosinophilic, large) Major basic protein (attacks parasite larva) Eosinophilic cationic protein Neurotoxin (cytotoxic, anti-RNA viral, chemoattractant) Histaminase (degrades histamine) Non-specific granules (lysosomes, azurophilic, large, fewer than PMN) Lysozyme (digests bacterial cell wall) Acid hydrolase (>50 enzymes that work at low pH) Myeloperoxidase (antimicrobial)  Basophils Anti-inflammatory Degranulation Upon IgE recognition Release of histamine induces vasodilation anaphylactic shock Chemotactic eosinophil chemotactic factor recruits eosinophils Induce and maintain allergic reactions Similar actions to Mast cells  Basophils 50 per microliter Days to years? 15-20 micron diam. Large irregular nucleus Nucleus obscured Clear cytoplasm Numerous large purple granules Easily activated/degranulated  Basophils TEM of basophils shows intensely dark granules due to heparin content. Myelin figures are lipid membrane-like coils associated with granules that contain phospholipids for leukotriene synthesis.  Basophils Specific granules (very azurophilic, very large, numerous) Histamine (vasodilator) Heparin (anticoagulant, vasodilator) Eosinophil chemotactic factor Phospholipids for synthesis of leukotrienes Non-specific granules (lysosomes, less azurophilic, fewer) Lysozyme (digests bacterial cell wall) Acid hydrolase (>50 enzymes that work at low pH) Myeloperoxidase (antimicrobial)  Lymphocytes Mediate immune responses Recognize foreign antigens Recognize antibody-antigen interaction Direct killing of malignant cells Direct killing of virus-infected cells Small Lymphocytes (inactive) B-cells and memory B-cells T-cells (cytotoxic, helper, regulatory) Large Agranular Lymphocytes (active) B-cells T-cells Large Granular Lymphocytes Natural Killer (NK) cells  Lymphocytes 2000/microliter Days to years Small 10-18 micron diam. Condensed chromatin Large nucleus small-round Large large-irregular Agranular NK-irregular Cytoplasm clear to light blue granules in NK cells Large Granular Large lymphocytes are activated (NK)  Lymphocytes By TEM, the cytoplasm of most lymphocytes appears Small devoid of activity due to lymphocyte the lack of organelles. A few mitochondria and ribosomes are visible. In the NK cell, the large granules are visible and NK uniformly of moderate Cell density.  Lymphocytes Most activity Cell surface receptors MHC, TCR, selectins, integrins Cytosolic signaling events NK cells Granules Perforin puts holes in target cell osmotic collapse of target Granzyme enters target cell initiate apoptosis  Monocytes Perform immune surveillance Differentiate into macrophages upon tissue entry Diapedesis/Chemotaxis Recognition of inflammatory signals Populate tissues as resident macrophages Kupffer Alveolar Microglia Osteoclast Langerhans Phagocytosis of tissue debris and foreign organisms Ab, Complement and non-specific recognition Antigen presentation Cytokine secretion to recruit cells (smc, fibroblasts)  Monocytes 400/microliter Days to years 15-30 micron diam. Irregular nuclear shape (U) Chromatin moderately condensed Gray-pink cytoplasm Small blue/purple granules Prominent clear vacuoles W-G staining identifier  Monocytes In TEM images of monocytes, the lysosomal granules appear dense, but the vacuoles seen in W-G stains are less prominent due to sample preparation.  Monocytes Vacuoles Membrane bound organelles Contain phagocytosed material Will fuse with lysosomes Lysosome Membrane bound organelles Contain acidic hydrolases Process antigen for presentation to lymphocytes Azurophilic  Thrombocytes(platelets) Hemostasis Form physical barrier to blood loss Integrate with protein (fibrous) clot Fibrinogen binding Retract wound edges cytoskeletal contraction Potentiate coagulation cascade Regulator of vascular tone Release of granule contents  Thrombocytes(platelets) 300,000/microliter 10 day lifespan Anucleate cell fragment 2-3 micron diam. Typically clumped Typically activated Overall azurophilia No discernible contents by W-G TEM reveals cortical cytoskeleton and granules of platelets.  Thrombocytes(platelets) Delta (Dense) granules (not visible) ADP, ATP (paracrine platelet activation, vasoconstriction) Serotonin (vasoconstrictor, stimulates fibrocytes) Alpha granules (not visible) Platelet Derived Growth Factor (PDGF, promotes wound remodeling by other cells) Thrombocytes(platelets) Hemostasis Fibrous Cellular clot  Peripheral Blood Scott D. Blystone, Ph.D.