1. Red Blood Cells, Anemia and Polycythemia Prof. dr. Zoran Valić Department of Physiology University of Split School of Medicine

2. Red Blood Cells (Erythrocytes)  functions: 1) transport of hemoglobin (O 2 )  in some animals it circulates as free protein  in humans within RBC – loss by filtration 3% 2) large quantity of carbonic anhydrase (CO 2 and H 2 O) 3) an excelent acid-base buffer (proteins)

4.  biconcave discs (φ=7,8 μm; V=90-95 μm 3 )  shape can change remarkably (squeeze through capillaries, excess of membrane)  M = 5,2x10 12  F = 4,7x10 12  c hemoglobin in RBC < 340 g/L  Ht = 40-45%  c hemohlobin in blood = 160-140 g/L

7. 1) yolk sac (few early weeks) 2) liver; spleen and lymph nodes(middle trimester of gestation) 3) bone marrow  beyond the age of 20 most RBC are produced in membranous bones (vertebrae, sternum, ribs and ilia)

10.  growth inducers – proteins which control growth and reproduction of stem cells  interleukin-3 – promotes growth and reproduction of virtually all stem cells  differentiation inducers (low oxygen, infectious diseases)

11.  1% bone marrow

13.  tissue oxygenation – most essential regulator (viscosity)  hemorrhage, x-ray therapy, high altitudes, cardiac failure, lung diseases  erythropoietin (glycoprotein; 34000)  90% is formed in kidneys (unknown, liver)  fibroblast-like interstitial cells surrounding the tubules?  renal tissue hypoxia (and some other)   HIF-1   erythropoietin  quick secretion (min – 24 h), RBC in 5 days  production of proerythroblasts, speeding up

14.  erythropoietic cells are among the most rapidly growing and reproducing cells  person’s nutritional status  vitamin B 12 and folic acid (thymidine)  macrocytes – flimsy membrane and irregular, large shape – shorten life span (1/2-1/3 normal)  B 12 – pernicious anemia (atrophic gastric mucosa; parietal cells – intrinsic factor)  folic (pteroylglutaminic) acid – widely spread but destroyed during cooking – sprue

15. Formation of Hemoglobin  begins in proerythroblasts and continues even into the reticulocyte stage  succinyl-CoA from Krebs metabolic cycle  alpha, beta, gamma and delta chains  most common – hemoglobin A (2 alpha, 2 beta chains)  each hemoglobin molecule transports 4 molecules of oxygen

19.  sickle cell anemia –the amino acid valine is substituted for glutamic acid at one point in each of the two beta chains  15 μm elongated crystals in low oxygen environment  loosely and reversibly combining with O 2  “coordination bond”, molecular oxygen

21. Iron Metabolism  hemoglobin, myoglobin, cytochrome- oksidase, peroxidase and catalase  total iron in the body – 4-5g (65% in hemoglobin, 4% in myoglobin, 15-30% in reticuloendothelial system and liver parenchymal cells)

23.  transferrin molecule binds strongly with receptors in the cell membrane s of erythroblasts in bone marrow – endocytosis  inadequate quantities of transferrin – failure to transport iron to the erythroblasts – hypochromic anemia

24. Absorption of Iron  liver secretes moderate amounts of apotransferrin into the bile – transferrin (with the iron, pinocytosis into enterocyts, plasma transferrin)  absorption is slow and limited; total body iron is regulated mainly by altering the rate of absorption

25. Life Span of RBC  average circulating time  120 days  cytoplasmic enzymes: 1) maintaining pliability of the cell membrane 2) maintain membrane transport of ions 3) keep the iron in ferrous, rather than ferric form 4) prevent oxidation of the RBC proteins  many RBC self-destruct in the spleen (when squeezing through the red pulp)

26.  hemoglobin is phagocytized by macrophages (Kupffer cells of the liver)  iron and bilirubin (from porphyrin portion)

27. Anemias (deficiency of hemoglobin) 1) microcytic hypochromic anemia – blood loss anemia (acute and chronic) 2) aplastic anemia – bone marrow aplasia (high-dose radiation, chemotherapy, drugs, toxic chemicals – insecticides or benzene) 3) megaloblastic anemia (lack of B 12 (pernicious) or folic acid)

28. 4) hemolytic anemia (abnormalities (hereditary) of RBC) 1) hereditary spherocytosis (small and spherical RBC) 2) sickle cell anemia (hemoglobin S, crisis) 3) erythroblastosis fetalis

29. Effects of Anemia on Circulation  viscosity of blood depends largely on RBC  fall in blood viscosity  decrease in total resistance (added tissue hypoxia – vasodilation)  increase in CO (3-4x)  increased pumping workload on the heart  problems during exercise – acute cardiac failure

30. Polycythemia  secondary polycythemia – due to hypoxia (at high altitude, cardiac failure) – 6-7 x 10 12 (30%)  polycythemia vera (erythremia) – 7-8 x 10 12 (Ht = 60-70%) – genetic aberration in the hemocytoblastic cells  increased viscosity – CO almost normal (decreased venous return, but increased blood volume), ruddy complexion with a bluish (cyanotic) tint to the skin)

31. Blood Types; Transfusion; Tissue and Organ Transplatation

32. Antigenicity  first attempts were unsuccessful  transfusion reaction and death  blood posses antigenic and immune properties  at least 30 commonly occurring, and hundreds of other antigens  most of antigens are week, used to establish parentage  systems: O-A-B and Rh

33.  OAB system is discovered by Austrian scientist Karl Landsteiner 1900. (three types, awarded Nobel prize 1930; simultaneously with Czech serologist Jan Janský)  also with Alexander S Wiener identified Rh factor 1937.

34. O-A-B Blood Types  antigens A i B (also called agglutinogens – cause blood cell agglutination) occur on the surface of the RBC  because of the way of inheritance people may have neither of them on their cells, they may have one or they may have both simultaneously

35.  when neither A or B agglutinogen is present – blood (person) is blood type O  only agglutinogen A – blood is type A  only agglutinogen B – blood is type B  when both agglutinogens are present – blood is type AB

37.  antigen H – essential precursor of OAB blood antigens  located on chromosome 19, posses 3 exons which are coding enzyme fucosyltransferase  enzyme creates H antigen on RBC  carbohydrate chain: β-D-galactose, β -D-N- acetilglucosamine, β -D-galactose i α-L- fucose (connection with protein or ceramid)

38.  OAB locus is on chromosome 9, has 7 exons  exon 7 is the biggest and contains the greatest portion of coding sequence  OAB locus has 3 allele types: O, A, B

39.  allele A codes glycosyltransferase which bindes N-acetylgalactosamine on D- galactose end of H antigen  allele B codes glycosyltransferase which bindes α -D-galactose on D-galactose end of H antigen  allele 0 has deletion in exon 6 – loss of enzimatic activity – only H antigen is present

41.  Relative Frequencies of the Different Blood Types: 047% A41% B9% AB3%  there are 6 different allele types among white population: (A1, A2, B1, O1, O1v i O2), in Asian population B type is more frequent

43. Agglutinins  antibodies directed at agglutinogens  immediately after birth – not present  they are formed 2-8 month after the birth  maximum titer is reached 8-10 years of age  gamma-globulins (IgM i IgG)  why are they produced?  environmental antigens (bacteria, viruses, plants, foods)

45.  for anti-A agglutinins – influenza  for anti-B agglutinins – gram-negative bacteria (E. coli)  “light in the dark” theory – viruses during replication process incorporate parts of host membrane

46. Agglutination Process  agglutinins have 2 (IgG) or 10 (IgM) binding sites for agglutinogens  attaching to two or more RBC – bounding together (clump of cells) – agglutination  plugging of small blood vessels throughout the circulation – physical distortion of the cells or phagocytosis – hemolysis of the RBC

47. Acute Hemolysis  on rare occasion  hemolysis occurs immediately in circulating blood  activation of the complement system – release of proteolytic enzymes (the lytic complex) – rupture of the cell membranes (existence of high titer of IgM antibodies – hemolysins)

48. Blood Typing  blood typing and blood matching  RBC are separated from the plasma and diluted with saline; mixing with anti-A and anti-B agglutinins

51. Rh Blood Types  spontaneous agglutinins almost never occur (difference)  person must first be massively exposed (transfusion)  six common types of Rh antigens (C, D, E, c, “d”, e; one of each pair in every person)  most prevalent is type D antigen (Rh +)  about 85 percent of white people are Rh +

52.  in reality two genes: RHCE i RHD  proteins which carry Rh antigens are transmembranic proteins (ion channel?)  RHD gene codes RhD protein with D antigen (on chromosome 1p)  RHCE gene codes RhCE protein with C, E, c, e antigens  there is no d antigen, “d” means lack of D antigen

55. Rh Immune Response  maximum concentration of anti-Rh agglutinins develop about 2 to 4 months after transfusion  delayed, mild transfusion reaction  erythroblastosis fetalis (mother Rh -, father Rh +, child inherits Rh from father; mother develops agglutinins for Rh which diffuse through the placenta into the fetus and cause red blood cell agglutination)

56.  firstborn usually doesn’t develop, second born in 3%, third born in 10%  agglutination of the fetus's blood – hemolysis – release of hemoglobin (jaundice)

57.  newborn baby is usually anemic, liver and spleen become greatly enlarged, early forms of RBC are passed from the baby's bone marrow into the circulatory system, permanent mental impairment or damage to motor areas of the brain because of precipitation of bilirubin in the neuronal cells – kernicterus

58.  treatment – replacing the neonate's blood with Rh-negative blood (400 ml during 1,5 hours)  RBC are replaced by infant's own at the time anti-Rh agglutinins that had come from the mother are destroyed

59. Prevention of Erythroblastosis Fetalis  development of Rh immunoglobulin globin, an anti-D antibody  administered to the expectant mother starting at 28 to 30 weeks of gestation or after delivery

60. Transfusion Reactions  usually agglutination of the RBC from the donor, rarely agglutination of cells in recipient (dilution of plasma)  hemolysis (immediate – hemolysins, later – phagocytosis)  jaundice (more than 400 milliliters of blood is hemolyzed in less than a day)

61.  acute kidney failure 1) release of toxic substances – renal vasoconstriction 2) loss of circulating RBC – circulatory shock 3) hemoglobin precipitates and blocks many of the kidney tubules 4) patient dies within a week to 12 days

62. Transplantation of Tissues and Organs  beside RBC antigens each tissue posses additional set of antigens which are responsible for immunological reactions  resisting invasion by foreign bacteria or red cells

63. Type of Transplant  autograft – tissue or whole organ from one part of the same animal to another part  isograft – from one identical twin to another  allograft – from one human being to another or from any animal to another animal of the same species  xenograft – from a lower animal to a human being or from an animal of one species to one of another species

64.  xenografts – immune reactions almost always occur, causing death of the cells in the graft within 1 day to 5 weeks after transplantation  skin, kidney (5 to 15 years), heart, liver, glandular tissue, bone marrow, and lung

65.  most important antigens for causing graft rejection are a complex called the HLA antigens (6 of these antigens are present on the tissue cell membranes of each person, but there are about 150 different HLA antigens to choose from – more than a trillion possible combinations; on the white blood cells, as well as on the tissue cells – tissue typing)

66. Prevention of Graft Rejection  suppressing the immune system  T cells are mainly the portion of the immune system important for killing grafted cells 1) glucocorticoid hormones and similar drugs 2) drugs that have a toxic effect on the lymphoid system – azathioprine 3) cyclosporine – specific inhibitory effect on the formation of helper T cells  infectious disease, incidence of cancer!