Disorders of Red Blood Cells Professor Myat Thandar Department of Physiology University of Medicine 1

Functions of RBCs O2 transport (Hb in the RBCs) CO2 transport Acid-base balance

Functional Importance of the Biconcave Shape of RBCs Larger surface area for O2 diffusion Thinness of cell membrane enables O2 to diffuse easily Flexibility of membrane facilitates the transport function

Network of Fibrous Proteins of RBCs Spectrin and Ankyrin Imparts elasticity and stability to membrane and allows RBCs to deform easily

Haemoglobin A natural pigment, reddish when oxygenated 4 polypeptide chains (a globin portion and a heme unit)

Haemoglobin F in Fetus Higher affinity for O2 than adult Hb HbF is replaced within 6 months of birth with HbA

Haemoglobin Synthesis Availability of iron for heme synthesis Amount of iron: 2 g in women and 6 g in men Clinically, decreased ferritin levels usually indicate the need for prescription of iron supplements.

Red Cell Production Until 5, almost all bones; After 20, membranous bones Approximately 1% of total RBC is generated from bone marrow each day Reticulocyte count serves as an index of erythropoietic activity of bone marrow

Stages of Erythropoiesis Hematopoietic stem cell (HSCs) Unipotent committed stem cell Proerythroblast (15-20 mm) Early normoblast (12-16 mm) Intermediate normoblast (10-14 mm) Haemoglobinization begins Late normoblast (10-14 mm) Haemoglobinization ++ Nuclear disintegration Reticulocyte (7-8 mm) Nucleus remains only as strands of reticular element Erythrocyte (7.5 mm) IL-1, IL-6, IL-3 (interleukins) GM-CSF, G-CSF, SCF Erythropoietin GM-CSF

Red Cell Production

Red Cell Maturation Reduction in the cell size Increase in the amount of haemoglobin Disappearance of nucleus, and Change in staining characteristics of cytoplasm: basophilic to eosinophilic. This is partly due to a fall in content of RNA.

Erythropoietin

Human Erythropoietin Produced by recombinant DNA technology Used for anaemia induced by chemotherapy in cancer patients, and HIV infected persons treated with zidovudine In severe anaemia, retic count may be as much as 30% (normal about 1%); numerous erythroblasts may appear in the blood

Destruction of Red Blood Cells

Excretion of Bilirubins Excess bilirubin elimination leads to bilirubin gallstones If red cell destruction and bilirubin production is excessive, yellow discoloration of the skin, jaundice, occurs due to accumulation of unconjugated bilirubin

Haemoglobinuria Haemoglobin binding protein – Haptoglobin – in the plasma Other plasma proteins – albumin – also binds to Hb Extensive destruction of RBCs (haemolytic transfusion reactions), binding capacity is exceeded Haemoglobinaemia and haemoglobinuria results

Red Cell Metabolism 2,3-DPG decreases affinity of Hb for O2, facilitating the release of O2 at tissue levels Increased 2,3-DPG occurs in chronic hypoxia such as chronic lung diseases, anemia and residence at high altitude

Inhibition of Oxygen Haemoglobin Binding Certain chemicals : nitrates and sulfates Hb reacts with nitrite to form methaemoglobin G6PD deficiency predisposes to oxidative denaturation of hemoglobin with resultant red cell injury and lysis (oxidative stress generated by infection or exposure to certain drugs)

Laboratory Tests Using automated blood cell counters: red cell content and indices Red cell indices are used to differentiate type of anemias by size or color of red cells Haemoglobin Hematocrit Mean corpuscular volume (MCV falls in microcytic and rises in macrocytic anemia) Mean corpuscular haemoglobin concentration (normochromic or normal MCHC; hypochromic or decreased color or decreased MCHC)

Laboratory Tests Mean cell haemoglobin A stained blood smear: information about size, color and shape of red cells and the presence of immature or abnormal cells If blood smear is abnormal, bone marrow examination may be indicated Bone marrow aspiration from posterior iliac crest or the sternum

Red cell count and Haemoglobin severity of anemia Red cell characteristics Size normocytic, microcytic or macrocytic Color normochromic, hypochromic Shape the cause of anemia

Anemia Values of hemoglobin, hematocrit or RBC counts which are more than 2 standard deviations below the mean HGB<13.5 g/dL (men) <12 (women) HCT<41% (men) <36 (women) Normal Hb Concentration Western value Myanmar value Male : 16 g / dL (14 - 17 g/dL) 14.4 g / dL Female : 14 g / dL (12 - 15.5 g/dL) 12.5 g / dL

Pathophysiology of Anemia Blood Loss Decreased Production (lack of nutritional elements or bone marrow failure) Increased Destruction (haemolysis)

Effects of Anemia Manifestations of impaired oxygen transport and the resultant compensatory mechanisms Reduction in red cell indices and hemoglobin levels Signs and symptoms associated with the pathophysiologic process that causes anemia Manifestations depend on its severity, the rapidity of its development and the person’s age and health status

Symptoms of Anemia

Impaired oxygen transport and tissue hypoxia Weakness, fatigue, dyspnoea and angina Brain hypoxia results in headache, faintness and dim vision Redistribution of blood results in pallor of skin, conjunctiva, mucous membranes and nail beds

Compensatory Mechanisms Tachycardia, palpitations and increased cardiac output A flow type of systolic murmur Ventricular hypertrophy and high output heart failure Accelerated erythropoiesis results in diffuse bone pain and sternal tenderness Haemolytic anemia : jaundice Aplastic anemia : petechiae and purpura due to reduced platelet functions

Blood Loss Anemias Depends on rate of haemorrhage and blood loss is external or internal Rapid loss causes circulatory shock and collapse; fall in red cell count, Hb, hematocrit due to fluid shift into vessels Initially red cells are normocytic, normochromic Increased erythropoietin and retic count Slow loss (GI bleeding, menstrual disorders) causes anemia; signs and symptoms develop if the amount of red cell mass loss reach 50% (Hb <8 g/dL)(iron deficiency anemia) External bleeding leads to iron loss and iron deficiency

Haemolytic Anemias Characterized by premature destruction of red cells, retention in the body of iron and other products of Hb destruction and increased erythropoiesis Normocytic normochromic red cells Increased retic count in the circulating blood Haemoglobinemia, haemoglobinuria, jaundice, haemosiderinuria

Haemolytic Anemias Intravascular haemolysis is less common, caused by complement fixation in transfusion reactions, mechanical injury or toxic factors Extravascular haemolysis occurs when RBCs are less deformable to traverse splenic sinusoids, characterized by anemia and jaundice Intrinsic : defects of red cell membrane, haemoglobinopathies (sickle cell disease and thalassemias) and enzymes defect Extrinsic or acquired : drugs, bacteria and other toxins, antibodies and physical trauma

Inherited Disorders of Red Cell Membrane Hereditary spherocytosis : abnormalities of spectrin and ankyrin Mild hemolytic anemia, jaundice, splenomegaly and bilirubin gallstones Splenectomy done to reduce red cell destruction and blood transfusion in a crisis

Sickle Cell Disease Haemoglobin S (point mutation in the β chain of Hb, valine for glutamic acid) Haemolytic anaemia, jaundice, gallbladder stones, pain and organ failure (infarction of organs) Hb S becomes sickled when deoxygenated or at a low oxygen Deoxygenated Hb aggregates and polymerizes in the cytoplasm, creating a semisolid gel that changes the shape and deformability of the cell

Red Cell Sickling Chronic hemolytic anemia Blood vessel occlusion Associated conditions: cold, stress, physical exertion, infection, illnesses that cause hypoxia, dehydration or acidosis

Features of Sickle Cell Disease

Diagnosis and Treatment Neonates : clinical findings and haemoglobin electrophoresis Prenatal diagnosis : analysis of fetal DNA by amniocentesis Prevention of sickling episodes, symptomatic treatment and treatment of complications (prophylactic penicillin and full immunization) Cytotoxic drug – hydroxyurea – to allow synthesis of more HbF and less HbS Nitric oxide appears to be a promising new drug Bone marrow or stem cell transplantation

Thalassemias Inherited disorders of haemoglobin synthesis and decreased synthesis of α or β globin chains of HbA Heterozygous or homozygous

Thalassemias β thalassemias – Cooley anaemia or Mediterranean anemia – common in Mediterranean population of southern Italy and Greece α thalassemias more common among Asians Anemia due to low production of affected chain and continued production and accumulation of unaffected globin chain Reduced Hb synthesis leads to hypochromic microcytic anemia; accumulation of unaffected chain interferes with normal red cell maturation, and membrane changes leading to hemolysis and anemia

β - thalassemias Excess α chains are denatured to form precipitates (Heinz bodies) in the bone marrow red cell precursors Heinz bodies impair DNA synthesis and damage to red cell membrane Coagulation abnormalities, thrombotic events (stroke and pulmonary embolism) in moderate to severe form

Pathophysiology of β - thalassemias Thinning of cortical bones

Treatment of β - thalassemias Regular transfusion to maintain Hb at 9 to 10 g/dL Iron chelation therapy to reduce iron load Stem cell transplantation Stem cell gene replacement

α - thalassemias Synthesis of globin chains is controlled by 4 genes Deletion of single gene : silent carrier; two genes is α thalassemia trait Deletion of three genes leads to unstable aggregates of α chains – HbH Four globin chains are deleted : Hb Bart (extremely high oxygen affinity, cannot release oxygen in the tissues Chronic hemolytic anemia

Inherited Enzymes Defect G6PD deficiency RBCs vulnerable to oxidants, direct oxidation of Hb to methaemoglobin, and denaturation of Hb to form Heinz bodies Anti-malaria drug primaquine, the sulfonamides, nitrofurantoin, aspirin, phenacetin, some chemotherapeutics and other drugs cause hemolysis Diagnosed through G6PD assay or screening test

Acquired Hemolytic Anaemias By direct membrane destruction or antibody mediated lysis Various chemicals, toxins, venoms, malaria infection, prosthetic heart valves, vasculitis, severe burns, septicaemia, thrombotic thrombocytopenic purpura, renal disease Warm reacting antibodies (IgG) and cold reacting antibodies (IgM) Warm antibodies bind with Ag on red cell membrane (Rh Ag), resulting in spherocytosis and destruction by RE system Cold antibodies activate complements; as in lymphoproliferative disorder and idiopathic

Rh incompatibility

Coomb’s Test Direct Antiglobulin Test (DAT) is positive in autoimmune hemolytic anaemia, erythroblastosis fetalis, transfusion reactions, transfusion reactions and drug induced hemolysis Indirect antiglobulin test is used for antibody detection and crossmatching before transfusion

Anemias of Deficient Red Cell Production Deficiency of nutrients for hemoglobin synthesis (iron) Deficiency of nutrients for DNA synthesis (Cobalamin or folic acid) Marrow is replaced by nonfunctional tissues

Iron Deficiency Anemia Dietary deficiency (vegetarians) Loss of iron through bleeding (peptic ulcer, polyps, cancer, menstrual bleeding) Increased demands (growing children, pregnancy)

Iron Metabolism

Characteristics of Iron Deficiency Anemia Low haemoglobin and hematocrit Decreased iron stores, low serum iron and ferritin Red cells number decreased and are microcytic and hypochromic Poikilocytosis (irregular shape) Anisocytosis (irregular size) Reduced MCHC and MCV Membrane changes predispose to hemolysis

Manifestations of Iron Deficiency Anemia

Treatment of Iron Deficiency Anemia Prevention and treatment of the cause Ferrous sulfate Parenteral iron (iron dextran or sodium ferric gluconate) Initial test dose to prevent severe anaphylactic reactions

Megaloblastic Anemia Impaired DNA synthesis Enlarged red cells (MCV >100 fL) Develop slowly Vitamin B12 and folic acid deficiency

Vitamin B12 Deficiency Anemia: B12 Absorption

Pernicious Anemia Atrophic gastritis Autoimmune destruction of gastric mucosa Gastrectomy, ileal resection, inflammation or neoplasms in terminal ileum, malabsorption syndrome MCV elevated; MCHC is normal

Vitamin B12 Containing Food Normal body stores of 1000 to 5000 µg provide the daily requirement of 1 µg for a number of years. Therefore, deficiency develops slowly

Diagnosis of B12 Deficiency The Shilling test – 24 hour urinary excretion of radiolabelled vitamin B12 administered orally Detection of parietal cell and intrinsic factor antibodies Lifelong intramuscular or high oral doses of vitamin B12 is required

Folic Acid

Folic Acid Deficiency Total body stores amount to 2000 to 5000 µg and 50µg is required in the daily diet. A dietary deficiency may result in anaemia in a few months Pregnancy increases the need for folic acid 5 to 10 fold

Aplastic Anemia Reduction of all 3 hemopoietic cell lines Onset may be insidious but may be abrupt and severe

Therapy in Aplastic Anemia

Therapy in Aplastic Anemia Immunosuppressive therapy with lymphocyte immune globulin Avoid offending agents Antibiotics for infection Red cell transfusion to correct anaemia Platelets and corticosteroid therapy to minimize bleeding

Chronic Disease Anemia Occur as a complication of chronic infections, inflammation, cancer and chronic kidney diseases Short red cell life span; deficient red cell production; a blunted response to erythropoietin, and low serum iron Mild anemia – normocytic and normochromic – with low reticulocyte counts In chronic renal diseases, uremic toxins and retained nitrogen interfere with actions of erythropoietin; hemolysis and blood loss associated with hemodialysis and bleeding tendencies also contribute to anemia

Therapy in Chronic Disease Anemia Short-term erythropoietin therapy Iron supplementation Blood transfusions In future – iron chelating agents and cytokines to stimulate erythropoietin production

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