Microbiology 435 Erythrocytes

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Erythrocytes are also known as red blood cells, erythrocytes, or red corpuscles. Then term erythron refers to all the erythrocytes and their precursors in the blood, bone marrow or at extramedullary sites. Erythrocytes transport oxygen and carbon dioxide between the lungs and all the tissues of the body. A circulating erythrocyte is little more than a container for hemoglobin. Characteristics of individual Erythrocytes diameter = 6 to 8 microns thickness = 1.5-1.8 microns (a biconcave disk) production rate = 2.5 million/sec. or 200 billion/day life span = approximately 120 days Erythropoiesis is the development of erythrocytes. Sites of development Erythropoietic tissue originates in the yolk sac then moves to the liver and spleen during fetal life. Eventually erythropoiesis settles in the medullary cavity of the skeleton. By about 18 years of age the axial skeleton and proximal ends of the long bones are the site of erythrocyte production. Nutrients needed for effective erythropoiesis amino acids (proteins)- for globin production iron - for heme production vitamins B12, B6, and folic acid (part of B2) nickel and cobalt as trace minerals Erythropoietin (EPO) Erythropoietin is a hormone that controls erythropoiesis. Its production and release is governed by: the need to keep the normal oxygen carrying capacity of the blood at a steady state based on the normal turnover of erythrocytes. the oxygen content of tissues (hypoxia leads to increased production). Erythropoietin acts primarily at the CFU-E stage of committed erythroid cells. It also acts at the BFU-E stage to a lesser degree. Stages of Erythropoiesis Erythropoiesis starts with the pluripotent stem cell as does all hematopoiesis In the presence of the proper growth factors the pluripotent stem cell differentiates into the CFU-GEMM. Partially under the influence of EPO the CFU-GEMM differentiates into a BFU-E. The BFU-E is considered the earliest cell in the erythrocyte series, even though we will not be able to identify it on a bone marrow biopsy or a peripheral blood smear. The BFU-E then differentiates into the CFU-E. The CFU-E is heavily under the influence of erythropoietin and will differentiate into the identifiable cells in the erythrocyte cell line. Erythropoiesis - general overview of cellular changes Rubriblast Prorubricyte Rubricyte Metarubricyte Reticulocyte Erythrocyte Cell volume decreases as the cell matures. On the average the size goes from rubriblast measuring 12-19 microns to a mature erythrocyte measuring 6-8 microns in diameter. Chromatin condenses. The rubriblast has very fine chromatin; the metarubricyte has solid chromatin and the mature erythrocyte has none. Nucleoli disappear by the rubricyte state. Nuclear shape remains round. N:C ratio decreases. The rubriblast and prorubricyte have an N:C ratio of 4:1; the rubricyte and metarubricyte N:C ratio is 1:1. RNA activity decreases in cytoplasm resulting in lighter blue cytoplasm as the cell matures. The rubriblast cytoplasm is deep blue, the rubricyte cytoplasm is pinkish blue, the pink coming from the beginning of hemoglobin production. Hemoglobin production begins at the rubricyte stage and increases as the cell matures. There is a gradual shifting of predominantly blue to predominantly pink cytoplasmic color as the cell matures to an erythrocyte. A mature erythrocyte has no blue color in the cytoplasm. Mitochondria activity decreases. You may see a halo around the nucleus in the rubriblast and prorubricyte. The nucleus is eventually extruded. The metarubricyte is last stage with a nucleus. Blue color of cytoplasm due to presence of RNA indicating protein synthesis. Pink color of cytoplasm due to presence of hemoglobin production. Perinuclear halo indicates mitochondria and Golgi apparatus surround the nucleus. These structures do not pick up stain. From 14 to16 erythrocytes are produced from one rubriblast. Erythrocyte maturation times Rubriblasts usually divide within 12 hours of stimulation to make 2 daughter cells (prorubricytes) Prorubricytes require about 20 hours to develop. The rubricyte stage lasts about 30 hours. A metarubricytes last about 48 hours. Reticulocytes are released from bone marrow after 2 to 3 days and circulate for an additional 1 or 2 days before maturing into an erythrocyte. A mature erythrocyte lives about 120 days. Composition of Bone Marrow rubriblasts = 1% or less prorubricytes = 1 to 4% rubricytes = 10 to 20% metarubricytes = 5 to 10% WBC:nucleated erythrocyte ratio is 4:1 (WBCs have shorter life span than erythrocytes therefore we have a larger storage (reserve) pool of WBCs in case of emergency.  Reticulocytes About 1% of the circulating erythrocyte mass is generated by the marrow each day so that on a given day there should be 1% reticulocytes in the peripheral blood. These replace the 1% of the erythrocyte mass that dies of old age each day. The reticulocyte is an immature erythrocyte. It lacks a nucleus but contains various organelles (mitochondria and ribosomes) and still doesn’t have its full compliment of hemoglobin. If stained with a supravital stain such as New Methylene Blue, the remaining RNA in the reticulocyte is precipitated by the stain to form a mesh-like pattern. With Wright’s stain the reticulocytes may appear polychromatic (blue/pink cytoplasm), depending on the amount of remaining RNA. The rate of reticulocyte release from the marrow into the peripheral circulation is governed primarily by the rate at which O2 is being supplied to the tissues. A decrease in PO2 (hypoxia) is recognized by the kidney, which is stimulated to release erythropoietin. Hemoglobin Hemoglobin is a conjugated globular protein that constitutes 33% of the erythrocyte’s weight by volume. Its function is to carry O2 from the lungs to the tissues and carry CO2 from the tissues to the lungs. Hemoglobin has a molecular weight of 68,000 making it a large protein. Each normal erythrocyte contains 200-300 million molecules of hemoglobin. 65% of hemoglobin synthesis occurs during the nucleated stages of maturation and 35% during the reticulocyte stage. Normal values male: 13.5 to 18 g/dl female: 11.5 to 16 g/dl Composition of hemoglobin: Each hemoglobin molecule consists of 4 heme groups and 1 globin molecule. Each heme group contains a protoporphyrin ring plus an iron molecule. Each globin consists of 4 polypeptide chains (2 pairs). The synthesis of protoporphyrin and globin is coordinated, so if production of one decreases the production of the other also decreases. Globin formation The polypeptide chains of globin are produced on the ribosomes. The four most common chain types are alpha, beta, gamma and delta chains. Each of these chains differs from the others in their amino acid sequence. Each globin molecule is made of 2 pairs of chains and each chain is made of 141-146 amino acids. Heme formation Heme formation takes place in the mitochondria then the cytoplasm of erythroid precursors through the reticulocyte stage. It begins with production of a protoporphyrin ring. Iron then incorporates with protoporphyrin to form heme. Porphyrias are a group of disorders that have specific enzymatic problems in the chemical pathway leading to heme production. The most common is acute intermittent porphyria characterized by colicky abdominal pain, vomiting, diarrhea, fever, increased WBCs, and increased blood pressure. Any part of the nervous system can be affected and can result in CNS changes like behavioral changes and frank psychosis. Heme synthesis is stimulated by EPO (erythropoietin) and inhibited by the presence of heme. Configuration The polypeptide chains of adult hemoglobin are organized into 2 alpha chains and 2 beta chains; each chain has an attached heme group. In this configuration hemoglobin carries its maximum amount of O2; if the hemoglobin molecule is denatured (altered) it looses its ability to carry O2. A complete hemoglobin molecule is spherical, has 4 heme groups attached to the 4 polypeptide chains so it may carry up to 4 molecules of O2. Heme is suspended between portions of a polypeptide chain. Normal hemoglobin types embryonic hemoglobin is found in developing fetuses until approximately 12 weeks of age. It has a particular chain composition and is a very primitive hemoglobin. fetal hemoglobin (hemoglobin F) is present in fetal blood during the 5th week through the 9th month of gestation and after birth. The switch to adult hemoglobin in not complete for 3 to 6 months. Hemoglobin F functions very well in the low O2 environment in the uterus because its affinity for O2 is higher than normal adult hemoglobin. adult hemoglobin (hemoglobin A) is made of 2 alpha and 2 beta chains. It makes up 95 to 97% of adult hemoglobin. Adult erythrocytes also contain hemoglobin A2 (2 alpha and 2 delta chains) and hemoglobin F. glycosylated hemoglobin is a subfraction of hemoglobin A. It’s of interest for monitoring glucose metabolism in diabetics because the formation of glycosylated hemoglobin depends on the glucose concentration in the body. The amount of glycosylation of hemoglobin A reflects the sugar metabolism of the body over several days and is more of a steady-state measure of glucose than is a regular serum glucose level that is subject to immediate diet, exercise and other factors. Variant/Abnormal hemoglobins A. Hemoglobins with the oxygen-carrying capacity altered. In these hemoglobins a different molecule replaces the O2 molecule. carboxyhemoglobin - O2 has been replaced by CO (carbon monoxide). CO preferentially binds to hemoglobin over O2 by an affinity 210 times greater than O2. The blood is cherry red and the patient has a "healthy flush." HemoglobinCO is very stable but pure O2 administration does increase the odds of hemoglobin binding with O2. This is the leading cause of death by fire; people do not become cyanotic or experience respiratory distress. It kills "softly and quietly." sulfhemoglobin - sulfur has replaced the oxygen. This is also a stable form of hemoglobin It may result in denatured hemoglobin, which precipitates out as Heinz bodies. It is associated with administration of oxidizing drugs (certain antibiotics), Clostridium infection, severe constipation. Levels seldom reach 10% and this is usually not life threatening. methemoglobin is the oxidized state of hemoglobin with iron in the ferric state. Hemoglobin is unable to bind to O2 in this state. This is an inherited or acquired state. In cases of acquired methemoglobinemia if exposure to causative drugs or oxidant chemicals are removed, the methemoglobin will revert to normal hemoglobin. B. Abnormal hemoglobins from genetic alteration of the amino acid sequence. This condition is called a hemoglobinopathy and results in structural rearrangements of the hemoglobin molecule. Alteration has occurred during synthesis of one of the globin chains, e.g. in sickle cell disease there is a substitution of one amino acid on the beta chain for another amino acid. The sickled cells are not capable of normal oxygen transport. C. Abnormal hemoglobins from the altered rate of synthesis of one chain. These conditions are called thalassemias. A normal sequenced chain is produced but the decreased rate of synthesis results in disease, which in turn results in a decreased amount of hemoglobin produced. Any excess chains will form inclusions in the erythrocyte cytoplasm, which mark the cells for destruction by the macrophages. This also can be an inherited condition. D. Abnormal hemoglobins with no clinical or functional effect. Hundreds of these exist but do not result in a noticeable difference in hemoglobin function. Protein synthesis and production of hemoglobin in the various stages of erythropoiesis Rubriblast - Most of the iron to be used in hemoglobin synthesis is taken into the cell at this stage. The very blue cytoplasm, means protein (for hemoglobin) is being produced. Rubricyte - Hemoglobin appears for the first time. If any asynchrony between the development of the nucleus and the cytoplasm occurs, it’s first detected at this stage. Metarubricyte - The nucleus is extruded in later period of this stage. After this stage cell no longer can undergo mitosis Reticulocyte - Part of this stage occurs in the bone marrow, part in peripheral blood. At this stage the cell has approximately 2/3 of its total hemoglobin content. The cytoplasmic color at this stage is called polychromatic or polychromatophilic when viewed with Wright’s stain. If stained with new methylene blue, the remaining RNA will precipitate resulting in a reticular appearance. Mature erythrocyte - All the hemoglobin is now produced so there is no need for continued RNA and protein synthesis. These cells are pliable and deformable, making them capable of unusual changes in shape for passage through the microcirculation. Iron kinetics The source of iron used in heme production is ingestion. After begin absorbed into the bloodstream, the iron is transported by transferrin, a carrier protein, to the bone marrow where it is incorporated into heme molecules. Excess iron is stored in erythrocytes, liver, spleen, and the mononuclear phagocyte system (macrophages). The storage forms of iron are ferritin and hemosiderin. Sideroblasts are erythrocytes with iron stores that are visible when stained with Prussian Blue stain. Tests for evaluation of iron serum ferritin - soluble storage form of iron that is proportional to iron stored in tissues serum iron - iron available in the serum for erythrocyte production. This test isn’t as closely related to body stores as is ferritin. total iron binding capacity (TIBC) - a measure of transferrin which is not bound to iron. The TIBC is inversely related to iron level. The percent saturation of transferrin is calculated by dividing the serum iron divided by the TIBC. This is a more accurate test than serum iron but not as accurate as ferritin. serum transferrin measured by EIA (enzyme immunoassay). Serum transferrin values correlate well with TIBC values. Heme breakdown During its 120 day life span the erythrocyte has traveled 200-300 miles. The process of aging is called senescence. Enzyme activity decreases (esp. glycolytic enzyme which helps break down glucose, the source of erythrocyte energy), and the cell looses its deformability. MCHC (mean corpuscular hemoglobin concentration) increases, the cell becomes rounder, and the MCV mean corpuscular volume) decreases. 90% of destruction of senescent Erythrocytes occurs by extravascular hemolysis. Macrophages of the mononuclear phagocyte system remove them from circulation. Macrophages of the spleen are especially active in removing aging, dead and abnormal erythrocytes (e.g. cells containing Heinz bodies or Howell-Jolly bodies, siderocytes, target cells, schistocytes, tear drop cells and antibody-coated erythrocytes). Some essential components of heme, (iron plus amino acids from globin of hemoglobin) are recovered from the macrophages. Iron is transported via transferrin to the red bone marrow. The amino acids from globin are returned to the liver where they are used to build more proteins. The protoporphyrin ring of heme is disassembled and from the body. Its alpha carbon is exhaled in the form of CO2. The opened tetrapyrrole, biliverdin, is converted to bilirubin which is then carried to the liver by the plasma protein, albumin. In the liver bilirubin is conjugated to glucuronide to make it water soluble and excreted along with bile into the intestines. In the intestines it is converted by bacteria into stercobilinogen and excreted in the stool; some is eliminated as urobilinogen in the urine. Stercobilinogen and urobilinogen are what give feces and urine their color. When there is excess erythrocyte breakdown, jaundice (icterus) results; seen as yellowness of the skin and sclera due to increased serum bilirubin and deposition of bile pigments. Unconjugated bilirubin (prehepatic) and conjugated bilirubin (posthepatic) are measured in serum as indirect (unconjugated) and direct (conjugated) bilirubin; used to monitor amount of hemolysis. Intravascular hemolysis About 10% of normal erythrocyte destruction occurs by intravascular hemolysis.  While in circulation the red cell is subjected to metabolic and mechanical stresses: turbulence, endothelial damage and fibrin deposition resulting in red cell fragmentation (schistocytes) and/or intravascular hemolysis. When the erythrocyte ruptures, hemoglobin is released into the blood. The hemoglobin dissociates into alpha-beta dimers and is picked up haptoglobin, a protein carrier, to prevent renal excretion of hemoglobin. Haptoglobin carries the hemoglobin to the liver for further catabolism where the process proceeds as with extravascular hemolysis. As haptoglobin is depleted, unbound hemoglobin dimers appear in the plasma (hemoglobinemia) and are reabsorbed by the kidney up to a certain level and converted to hemosiderin; beyond this level hemoglobin shows up in the urine (hemoglobinuria) Intravascular hemolysis results in pink, red or brown plasma (hemoglobinemia). Urine may also show red color (hemoglobinuria). Function of hemoglobin The major function of hemoglobin is the transport of oxygen to the tissues. There are two normal physiologic states of hemoglobin: oxyhemoglobin - hemoglobin is bound with oxygen, a O2 molecule is associated with each Fe++ molecule (ferrous iron). deoxyhemoglobin - hemoglobin is dissociated from oxygen. This is also known as empty hemoglobin. Factors such as pH, temperature, PO2 and PCO2 also influence the ability of the hemoglobin molecule to carry oxygen. Carbon dioxide transport Carbon dioxide is also transported by hemoglobin. CO2 transport from tissues can be accomplished by 3 mechanisms, directly and indirectly by erythrocytes and in solution in the plasma. Three-fourths of the CO2 is transported by indirect erythrocyte transport: CO2 diffuses into the erythrocytes where it is transformed into carbonic acid (H2O + CO2 = H2CO3 = H+ + HCO3–). Deoxyhemoglobin accepts the H+ and HCO3– (bicarbonate) diffuses back into the plasma. In the lungs HCO3– converts back to CO2 and H20 and is eliminated via respiration.