(III.) Erythrocyte sedimentation rate (IV.) Estimation of osmotic resistance of red blood cells Physiology I - practical Dep. of Physiology, Fac. of Medicine, MU 2015 © Michal Hendrych, Tibor Stračina Sedimentation of erythrocytes (RBCs) • Erythrocyte sedimentation rate (ESR) • Non-specific laboratory method, low sensitivity • Speed of the erythrocyte sedimentation in the column of anti-coagulated blood in a glass tube • The value of ESR is inversely related to suspension stability of blood Suspension stability of blood Helmholtz electrical double-layer • Negative charge on the membrane of red blood cell membrane • Due to attractive electrostatic forces, positive charged ions are driven • RBCs are driven away each from other RBC + - + + + + ++ + + + + + + + - - - - - - - - + + + + + - - - -- - - - - - - - - - - - - + Mechanism of erythrocyte sedimentation • Gravitational force • Factors impairing the Helmholtz electrical double-layer cause aggregation of RBCs into cylindrical aggregates (rouleaux) with higher volume and relatively smaller surface (compared to corresponding volume of separate RBCs) • Aggregates fall down (sedimentate) faster than separate RBCs = value of ESR is higher Basic factors affected ESR • Volume of RBC: bigger RBC falls down faster • Number of RBCs: higher RBC count decreases ESR • Plasmatic proteins • Albumin – is charged negatively • hypoalbuminemia = increased ESR • Immunoglobulins, fibrinogen – negative and positive charge, impairment of Helmholtz electric double-layer • Increased plasmatic concentration of immunoglobulins (e.g. due to systemic infection) = increased ESR Estimation of ESR • Fahraeus-Westergren (FW) • Glass tube in vertical position • Measured after 1 hour (2 hours) • Wintrobe • 100 mm long, thin glass tube in upright or oblique position • Measured after 15 minutes • Less sensitive than the FW Robin Fåhræus (1888-1968) Maxwell M. Wintrobe (1901-1986) Anti-coagulated blood • Blood sample with the coagulating system blocked • Possible anticoagulant agents • Chelation of Ca2+ ions (chelation anticoagulants) • Sodium citrate • EDTA (ethylendiaminetetraacetic acid) • Sodium oxalate • Activation of antithrombin III – heparin and its low molecular weight derivatives (LMWHs) Physiological values of ESR (FW) • Men: 2-8 mm/h • Women: 7-12 mm/h • Newborns: 2 mm/h • Infants: 4-8 mm/h • Intersexual differences in adults are caused by different RBC count and by differences in concentration of plasmatic proteins Changes of ESR (FW) • Increased ESR (FW) • Pregnancy, menstruation • Macrocythemia • Inflammation • Infection • Necrosis (myocardial infarction, injury) • Cancer • Relative/absolute loss of albumin (e.g. renal disorders) • Decreased ESR (FW) • spherocytosis • Polycythemia vera • Leukocytosis • Dysproteinemia – hypofibrinogenemia, hypogammaglobulinemia • Dehydration Diffusion • movement of molecules or atoms from a region of high concentration to a region of low concentration Filtration • Separation of fluids from the „solid“ parts through the membrane Osmosis • Flow of solvent (water) through semipermeable membrane according to osmotic gradient • Osmotic pressure – pressure necessary to stop the osmosis • Osmolarity – concentration of osmotic active particles in 1 liter of solution • Osmolality – concentration of osmotic active particles per 1 kg of solvent • Osmolality of plasma = 2*[Na+] + [glc] + [urea] = 275-295 mmol/kgH2O Tonicity • Osmolality of solution compared to osmolality of intracellular fluid • Hypotonic solutions • Isotonic solutions • 0.9% NaCl = physiological solution (saline) • Hypertonic solutions Hemolysis • Break-up of RBC due to disintegration of its membrane • Hemoglobin is spilled into the solution (plasma) • Mechanisms • Physical • Osmotic • Chemical • Immunological • Toxic Osmotic resistance of RBCs • Estimation of resistance of RBCs in hypotonic solutions • Specific method • differential diagnosis of hemolytic anemia • Minimal osmotic resistance – concentration of hypotonic solution of NaCl, in which first RBCs (minimal resistant) are broken • 0,4-0,44% • Maximal osmotic resistance – lowest concentration of hypotonic solution of NaCl, in which the small fraction of RBCs (maximal resistant) are still not broken • 0,3-0,33% • Osmotic resistance range – difference between minimal and maximal osmotic resistance Pathological values of osmotic resistance • Higher values of minimal osmotic resistance • Congenital hemolytic anemia • Lower values of maximal osmotic resistance • Polycythemia vera • Thalassemia • Sickle cell disease • Sideropenia • Status after splenectomy Sources of the pictures • Slide 6 - http://www.medipos.cz/odberove-nadoby-pomucky/pipeta-zkumcitrat-jednorazphdispette.html, http://www.coe.ou.edu/isb/awardees/1966.htm, http://www.hematology.org/Publications/Legends/3898.aspx, [cited 30.8.2015] • Slide 7 - http://www.chemicalland21.com/lifescience/foco/CALCIUM%20CITRATE.htm + https://cs.wikipedia.org/wiki/%C5%A0%C5%A5avelan_v%C3%A1penat%C3%BD#/media/File:Cal cium_oxalate.png + https://cs.wikipedia.org/wiki/Heparin#/media/File:Heparin-2D-skeletal.png [cited 30.8.2015] • Slide 10 - https://en.wikipedia.org/wiki/Passive_transport#/media/File:Scheme_simple_diffusion_in_cell_ membrane-en.svg [cited 30.8.2015] • Slide 11 - https://en.wikipedia.org/wiki/Passive_transport#/media/File:Filtration_diagram.svg [cited 30.8.2015] • Slide 12 - https://en.wikipedia.org/wiki/Osmosis#/media/File:0307_Osmosis.jpg [cited 30.8.2015] • Slide 13 - https://en.wikipedia.org/wiki/Passive_transport#/media/File:Osmotic_pressure_on_blood_cells _diagram.svg [cited 30.8.2015] • Slide 14 - http://labmed.ascpjournals.org/content/41/4/209/F2.expansion.html [cited 30.8.2015]