(I.) Red blood cell count. Estimation of haemoglobin concentration. Calculated parameters of red blood cells (II.) Estimation of blood group by slide method Physiology I – practicals Fyziologický ústav LF MU, 2015 © Michal Hendrych Red Blood Cell (RBC) – erytrocyte • Anucleated cell, the most abundant blood cell • Shape: • Biconcave disc • surface increased by 30% • spectrin • deformation inside the capillaries • Functions: • Transport of oxygen (haemoglobin) • Role in acidobasic balance and CO2 transport • Size: • Normocyte: 7.5 µm • Microcyte: ≤ 7 µm • Macrocyte: ≥ 9 µm • Megalocyte: ≥ 20 µm Reticulocyte • Immature RBC • In peripheral blood: 1% ± 0.5% of RBC • reticulocytosis: increased ratio of reticulocytes in peripheral blood • No nucleus, but residues of membrane organelles in cytoplasm (substantia granulo-filamentosa) • Within 48 hrs. maturation to RBC Alteration of shape: poicylocytosis Anisocytosis: varying size of RBCs RBC count • Number of RBCs • Men: 4.3-5.3 * 1012 / l • Women: 3.8-4.8 * 1012 / l • Newborns: 4.4-7.0 *1012 / l • Intersexual differences: • Men: testosterone (male sex hormone) stimulates releasing of erythropoietin • Women in fertile period: relative erythrocytopaenia due to menstruation Alterations of RBC count • polyglobulia – increased number of RBCs • erythrocytopaenia – decreased number of RBCs Estimation of RBC count • Automatic methods • Classical method: Bürker´s chamber + Hayem´s solution Haematocrit • Volume percentage of blood cells in blood sample (erythrocyte volume fraction) • Centrifugation of anti-coagulated blood • Plasma • Buffy coat • RBC • Hct (hematocrit) • Men: 42-52% • Women: 37-47% Haemoglobin • Haemoglobin concentration (HGB) • Men: 140-180 g/l • Women: 120-160 g/l • Newborns: 160-240 g/l Estimation of HGB • Spectrophotometric method: • Lysis of RBC by transforming solution + stabilisation of haemoglobin (Hb-cyanide) • Measurement of light absorption Haemoglobin - derivatives • Oxyhaemoglobin – haemoglobin + O2 • Carboxyhaemoglobin – haemoglobin + CO • Carbaminohaemoglobin – haemoglobin + CO2 • Methaemoglobin = haemoglobin with oxidized Fe (Fe3+) Calculated parameters of RBCs • Average volume of RBC (MCV, mean corpuscular volume) • MCV = HCT/RBC (hematocrit/ red blood count) = 80-95 fl • Average weight of Hb in RBC (MCH, mean corpuscular hemoglobin) • MCH = HGB/RBC (haemoglobin/ red blood count) = 28-32 pg • Average concentration of Hb in RBC (MCHC, mean corpuscular hemoglobin concentration) • MCHC = HGB/HCT (haemoglobin/ haematokrit) = 310-360 g/l • Red cell distribution width (RDW) = 11,5-14,5% • Variation of RBCs size • ↑RDW – anisocytosis Calculated parameters of RBCs HCT RBC HGB MCH = HGB/RBC MCHC = HGB/HCTMCV = HCT/RBC Anaemia • Decreased concentration of Hb in blood • Symptoms: • Pale mucose membrane • Fatigue • Tachycardia • Dyspnoe Sideropenic anaemia • Deficiency of Fe2+ → decreased production of RBCs→ hypoxia stimulates releasing of erythropoietin → increased production of RBCs with lack of Hb • Microcytic hypochromic anaemia Pernicious anaemia • Deficiency of B12 or folic acid • Macrocytic, hyperchromic anaemia Blood groups • Depend on antigens on the surface of RBC • Various systems: AB0, Rh, MNs, Kell, Lewis AB0 system • Agglutinogens – glycoproteins: • 0 – no agglutinogen, incidence: 33% • A – agglutinogen A, incidence: 45% • B – agglutinogen B, incidence: 16% • AB – both of agglutinogens A and B, incidence: 6% • Agglutinins – IgM antibodies: • 0 – anti-A + anti-B • A – anti-B • B – anti-A • AB – no agglutinin • Production of agglutinins starts in newborns AB0 system: heredity • Alleles: A, B, i • Co-dominancy: A and B • Allele i is recessive for both other alleles • Genotypes: • Group A: IA IA, IA Ii • Group B: IB IB, IB Ii • Group AB: IA IB • Group 0: Ii Ii Estimation of blood group by slide method • Slide method: • sera + small amount of blood • agglutination Agglutination vs. Coagulation Rh factor • Antigens C, c, D, d, E, e • D is determinative → Rh+, dominant • Rh- - recessive homozygote • Antibodies IgG – production after immunization by antigen D • Transfusion of incompatible blood • Delivery (miscarriage, abortion) of Rh+ fetus vs. Rh- mother • foetal erythroblastosis – hemolysis of foetal RBCs due to production of anti-D antibodies by maternal immune system → hydrops foetalis • Prevention: anti-D serum for mother after delivery/abortion – avert the immunization Picture sources • Slide 3 - https://en.wikipedia.org/wiki/Reticulocyte [cited 30.8.2015] • Slide 4 - http://medicaltreasure.com/poikilocytosis/ [cited 30.8.2015] • Slide 5 - https://commons.wikimedia.org/wiki/File:Anisocytosis.jpg [cited 30.8.2015] • Slide 7, 21 – Praktická cvičení z fyziologie, Masarykova univerzita 2011 • Slide 8 - http://www.wikiskripta.eu/index.php/Hematokrit [cited 30.8.2015] • Slide 9 – Ganong‘s Review of Medical Physiology, Ganong, McGrow Hill, 2010 • Slide 19 - http://www.wikiskripta.eu/index.php/Soubor:Krevni_skupiny.png [cited 30.8.2015]