HEMA1 - MIDTERM SAMPLE EXAM

Hemoglobin Metabolism

• Hemoglobin is an oxygen-transporting protein found in red blood cells (erythrocytes).
• The heme portion of hemoglobin gives red blood cells their characteristic red color.
• Hemoglobin's structure is a globular protein made of four heme groups and two different polypeptide chains.

Hemoglobin Structure

• X-ray crystallography was used to describe hemoglobin's structure for the first time.
• The molecule is a tetramer of four globin polypeptide chains, with one heme group imbedded in each chain.
• The four heme groups are positioned in a pocket of the polypeptide chain near the surface of the hemoglobin molecule.

Heme Structure

• Heme consists of protoporphyrin IX, a ring of carbon, hydrogen, and nitrogen atoms, with a central divalent ferrous iron (Fe2+) atom.
• Each heme group reversibly combines with one oxygen molecule, via the ferrous iron.
• When the ferrous iron oxidizes to the ferric state (Fe3+), it can no longer bind to oxygen.

Globin Structure

• Each hemoglobin molecule consists of four globin chains (two alpha chains and two beta chains) totaling 141 to 146 amino acids each.
• Variations in amino acid sequences result in different types of polypeptide chains.
• Each globin chain is designated by a Greek letter (alpha, beta, gamma, delta, epsilon, zeta, and theta).
• Each globin chain is divided into eight helices separated by seven non-helical segments.
• Flexible nonhelical segments connect the helices.
• The iron atom of heme is linked to the F8 proximal histidine on one side of the plane and oxygen binds to the iron atom on the other side, close to the E7 distal histidine.

Hemoglobin Molecule

• Hemoglobin's primary structure is the amino acid sequence of the polypeptide chains.
• The secondary structure describes how the chains are arranged in helices and non-helices.
• Globin chains loop to form a cleft pocket for the heme group.
• Each chain has a heme group that is suspended between the E and F helices of the polypeptide chains.
• Globin chain amino acids on the outside are hydrophilic, which makes the molecule water-soluble.
• Those in the cleft are hydrophobic, helping to keep the iron in a usable divalent ferrous form, whether the hemoglobin is oxygenated or deoxygenated.

Hemoglobin Synthesis

• 65% of hemoglobin is synthesized in immature red blood cells (reticulocytes).
• The remaining 35% is synthesized in reticulocytes.
• Heme synthesis occurs in the mitochondria of normoblasts and depends on glycine, succinyl coenzyme A, aminolevulinic acid synthetase, and vitamin B6.
• Globin synthesis occurs in ribosomes and is controlled on chromosome 16 for alpha chains and chromosome 11 for all other chains.
• Each globin chain binds to a heme molecule in the immature red blood cell cytoplasm.

Heme Synthesis

• Heme synthesis starts in mitochondria with the condensation of glycine and succinyl-CoA, catalyzed by aminolevulinic acid synthetase.
• This forms aminolevulinic acid (ALA).
• In the cytoplasm, ALA dehydratase (also known as porphobilinogen synthase) converts ALA to porphobilinogen.
• Further transformations occur, including hydroxylmethylbilane and coproporphyrinogen III development.
• The process continues in the mitochondria until, in the final step, Fe2+ combines with protoporphyrin IX in the presence of ferrochelatase (heme synthase) to form heme.

Globin Synthesis

The globin chains are synthesized from the pronormoblast stage through the polychromatic erythrocyte stage in the bone marrow.

• It involves DNA transcription in the nucleus to produce mRNA, which is then translated into polypeptide chains on ribosomes in the cytoplasm.
• The chains are released into the cytoplasm once complete.

Hemoglobin Ontogeny

• The z- and ε-globin chains appear only during the first three months of embryonic development by forming the embryonic hemoglobins.
• During the second and third trimesters of fetal life, and at birth, Hb F (α2γ2) predominates.
• Hb A (α2β2) becomes the predominant hemoglobin by six months of age and continues through adulthood, with small amounts of Hb A2 (α2δ2) and Hb F persisting.

Hemoglobin Types

• Fetal hemoglobin (Hb F) has alpha and gamma chains.
• Adult hemoglobin (Hb A) has alpha and beta chains.
• Adult hemoglobin A2 (Hb A2) has alpha and delta chains.
• Embryonic hemoglobins consist of specific combinations of zeta, epsilon, and gamma chains.

Hemoglobin/Erythrocyte Breakdown

• Intravascular hemolysis occurs when hemoglobin breaks down within the blood.
• Hemoglobin is released into plasma and binds to haptoglobin.
• This complex, along with hemopexin and albumin, is phagocytized by liver macrophages.
• Extravascular hemolysis occurs when senescent RBCs are phagocytized in the liver or spleen by macrophages.
• Protoporphyrin is metabolized to bilirubin and urobilinogen.
• Globin chains are recycled.
• Iron binds to transferrin and is transported to bone marrow for new red blood cell production, or it is stored as ferritin or hemosiderin.
• Intravascular hemolysis results in elevated plasma hemoglobin, serum bilirubin, and urine urobilinogen, and decreased serum haptoglobin.
• Extravascular hemolysis results in the same consequences, but with decreased plasma hemoglobin.

Hemoglobin and Iron

• Most body iron is in hemoglobin in its ferrous state (Fe2+), necessary for oxygen transport.
• Ferric iron (Fe3+) cannot bind to hemoglobin; it binds to transferrin.
• Serum iron (SI): measures the amount of Fe3+ bound to transferrin.
• Total iron-binding capacity (TIBC): measures the total iron-binding capacity of transferrin.
• Serum ferritin: measures stored iron in tissues and bone marrow.

Different Forms of Normal Hemoglobin

• Oxyhemoglobin: Hemoglobin with Fe²⁺ + O₂, found in arterial circulation.
• Deoxyhemoglobin: Hemoglobin with Fe²⁺ but no O₂, found in venous circulation.
• Carboxyhemoglobin: Hemoglobin with Fe²⁺ and carbon monoxide (CO). CO binds to hemoglobin more strongly than O₂.
• Methemoglobin: Hemoglobin with Fe³⁺; cannot transport oxygen and can result in cyanosis and anemia.
• Sulfhemoglobin: Hemoglobin with sulfur (S); cannot transport O₂; generally not fatal but caused by drugs and chemicals.

Oxygen Dissociation Curve

• Oxygen affinity is the ability of hemoglobin to bind or release oxygen.
• Expressed as the oxygen tension at which hemoglobin is 50% saturated with oxygen.
• The relationship between oxygen tension and hemoglobin saturation is described by the oxygen dissociation curve.
• Factors like the 2,3-DPG/BPG level, body temperature, and pH influence the curve, affecting oxygen release to tissues.

Disorders Related to Hemoglobin Biosynthesis

• Inherited defects include congenital erythropoietic porphyria.
• Acquired defects include lead poisoning (inhibits heme synthesis at multiple points).

Porphyria

• A disease of heme metabolism, marked by accumulation of porphyrins or precursors in the body.
• Classifications are based on clinical presentation (acute vs. chronic), the source of enzyme deficiency, and the site of enzyme deficiency in the heme biosynthetic pathway.
• Clinically, patients experience either neurological complications or skin problems. Some patients have no symptoms.

Porphyrin Synthesis Disorders

• Inherited defects involve a rare autosomal recessive condition called congenital erythropoietic porphyria.
• Acquired defects include lead poisoning, which inhibits heme synthesis at various points leading to abnormal reactions in the heme biosynthetic pathway.

Iron Metabolism Disorders

• Genetic variations affecting iron plasma concentrations are linked to conditions like transmembrane protease serine 6.
• Associated with lower hemoglobin levels, smaller red cells, and a high red blood cell distribution width (RDW).
• Primary overload disorders result from increased iron absorption from a normal diet.
• Secondary overload can arise from conditions like chronic disorders or hemolytic anemias, and iron therapy.
• Sideroblastic anemia involves excess iron accumulation in the cytoplasm of immature erythrocytes due to the ratio between plasma iron level and the iron required by the cell.
• Causes of sideroblastic anemia include congenital defects and acquired defects, such as primary (myelodysplastic syndrome) or secondary (drug-induced, toxin-induced) causes.

Erythrocyte Production and Destruction

• General characteristics of erythrocytes: biconcave shape, presence of hemoglobin for oxygen transport, loss of nucleus during maturation, limited lifespan (120 days).
• Erythropoiesis: The process of red blood cell production, beginning with hematopoietic stem cells (HSCs), transitioning through various stages (pronormoblast, basophilic normoblast, polychromatophilic normoblast, orthochromic normoblast, reticulocyte, erythrocyte) in the bone marrow.
• Regulation of erythropoiesis: Controlled partly by actions of cytokine signaling pathways and transcription factors, predominantly erythropoietin released in the kidneys.
• Erythrocyte function: Transport oxygen and removal of metabolic wastes.

Erythrocyte Physiology

• Energy production: Early red blood cells obtain energy via oxidative phosphorylation, which is later replaced by glycolysis as mitochondria are lost during maturation.
• Volume ratio and flexibility: Erythrocytes maintain a proper volume ratio to facilitate gas exchange through capillaries. The cation pump regulates the internal potassium (K+) and external sodium (Na+) ion concentrations to maintain flexibility.
• Membrane composition: The erythrocyte membrane is composed of approximately 50-60% lipids (phospholipids, cholesterol, and glycolipids) and 40-50% proteins.

Substances needed for erythropoiesis

• Iron (Fe²⁺): essential for oxygen transport.
• Amino acids: necessary for globin chain synthesis.
• Folic acid/vitamin B12: crucial for DNA replication and cell division.
• Erythropoietin,
• Vitamin 65 (pyridoxine): required for certain enzymatic reactions.
• Trace minerals: important for overall function.

Abnormal RBC distribution

• Rouleaux: Stacking or "coin-stacking" of erythrocytes due to abnormal or increased plasma proteins (e.g., in hyperproteinemia, multiple myeloma, Waldenstrom macroglobulinemia, or conditions with increased fibrinogen). • Agglutination: Clumping of erythrocytes without a specific pattern, occurring when erythrocytes are coated with IgM antibodies (e.g. in cold autoimmune hemolytic anemia).

Erythrocyte Morphology and Associated Diseases

• Normocytes: Normal size and shape (discocytes) of erythrocytes equivalent to the nucleus of a small lymphocyte.
• Macrocytes: RBCs larger than 8 µm in diameter (MCV >100 fL); seen in megaloblastic anemias, liver disease, or accelerated erythropoiesis.
• Microcytes: RBCs smaller than 6 µm in diameter (MCV <80 fL); seen in iron deficiency, thalassemia, sideroblastic anemia, or chronic disease.
• Anisocytosis: Variation in RBC size (dimorphism/heterogeneity). Common after treatment or post-transfusion or when multiple deficiencies are present(e.g. iron and B12).
• Poikilocytosis: Variation in RBC shape.
• Echinocytes (crenate or burr cells): Short, evenly spaced projections on the cell surface; often seen in liver disease, uremia, heparin therapy, pyruvate kinase deficiency.
• Acanthocytes (spur cells): Irregular, unevenly spaced projections on the cell surface; often seen in alcoholic liver disease, post-splenectomy, and abetalipoproteinemia.
• Target cells (codocytes or Mexican hat cells): Increased surface-to-volume ratio; central pallor area is surrounded by a ring; seen in liver disease, some hemoglobinopathies, thalassemia, iron-deficiency anemia.
• Spherocytes: Disk-shaped but small cells without central pallor; increased osmotic fragility; seen in hereditary spherocytosis, immune hemolytic anemia, and G6PD deficiency.
• Teardrop cells (dacryocytes): Pear-shaped cells often with a single blunt projection; seen in extramedullary hematopoiesis (e.g., myelofibrosis, myelophthisic anemia).
• Helmet cells (horn cells, keratocytes, schizocytes): Fragmented RBCs with a hollow interior; seen in microangiopathic hemolytic anemias like DIC, thermal injury, prosthetic implants.
• Schistocytes (RBC fragments): Small, irregular fragments of RBCs with pointed projections; common in microangiopathic hemolytic anemias, DIC, thermal injury.
• Stomatocytes (mouth cells): Elongated, slit-like central pallor; associated with osmotic changes due to altered cation balance (Na+ /K+); seen in liver disease, acute alcoholism, conditions affecting red cell membrane integrity.
• Elliptocytes (ovalocytes): Cigar- to egg-shaped cells, representing loss of red blood cell membrane integrity; common in hereditary elliptocytosis, iron deficiency anemia, and megaloblastic anemia.

Erythrocyte Inclusions and Associated Diseases

• Nucleated RBCs (nRBCs): Presence of nuclei in immature red blood cells; often indicate increased bone marrow activity.
• Howell-Jolly bodies: Small, round, DNA fragments; normally removed by the spleen; seen in sickle cell anemia, beta-thalassemia major, or other severe hemolytic anemias, megaloblastic anemia, or post-splenectomy.
• Basophilic stippling: Multiple, evenly dispersed, tiny inclusions in the cytoplasm; represent remnants of ribosomal RNA; often seen in thalassemia, megaloblastic anemia, lead poisoning, and alcoholism.
• Pappenheimer bodies: Small, irregular, dark-staining iron granules clustered at the cell periphery; found in sideroblastic anemia, hemoglobinopathies, and myelodysplastic syndromes.
• Cabot rings: Thin, red-violet ringlike structures, often in loop or figure-eight shapes; composed of fragmented nuclear material; seen in megaloblastic anemia, lead poisoning, and myelodysplastic syndromes.
• Hemoglobin C crystals: Condensed, intracellular, rodshaped crystals; found in hemoglobin C or SC disease.
• Hemoglobin SC crystals (Washington monument): Blunt, fingerlike projections; found in hemoglobin SC disease.
• Heinz Bodies: Small, denatured hemoglobin inclusions; insoluble hemoglobin fragments; seen in G6PD deficiency, beta-thalassemia major, and drug-induced anemia.

Malarial Parasites

• Microscopically visualizable erythrocytic inclusions and changes in cytoplasm due to different species of malaria.

Abnormal RBC Distribution and Related Diseases

• Rouleaux: Stacked or "coin-like" arrangement of erythrocytes due to abnormal or increased plasma proteins.
• Agglutination: A non-specific clumping of red blood cells; associated with cold autoimmunity hemolytic anemia.