Hemoglobin Variants Lecture Notes PDF

LECTURE | KATE CARLA RETES, RMT COLLEGE OF ALLIED MEDICAL SCIENCES DMC COLLEGE FOUNDATION, INC. HEMOGLOBIN VARIANTS o Oxygen affinity of the rema...

LECTURE | KATE CARLA RETES, RMT COLLEGE OF ALLIED MEDICAL SCIENCES DMC COLLEGE FOUNDATION, INC. HEMOGLOBIN VARIANTS o Oxygen affinity of the remaining heme groups increases (does not allow release of oxygen) Different hemoglobin that are not of normal form o Produces a SHIFT TO THE LEFT in the oxygen DYSHEMOGLOBIN dissociation curve dysfunctional hemoglobin that are unable to transport o Oxygen is not delivered efficiently to the tissues oxygen Methemoglobin levels >30% of total hemoglobin: hypoxia MODIFIED HEMOGLOBIN and cyanosis Elevated levels of methemoglobin may be seen in: Also known as Dyshemoglobin o Presence of oxidants Acquired hemoglobin variants whose structure has been o Decreased activity of methemoglobin reductase modified by drugs or environmental chemicals (genetic deficiency) 3 TYPES: ▪ NADH-methemoglobin reductase enzyme 1. METHEMOGLOBIN deficiency/diaphorase deficiency 2. SULFHEMOGLOBIN o Inherited Hb M disease (abnormality in the globin 3. CARBOXYHEMOGLOBIN portion of the hemoglobin molecule) METHEMOGLOBIN ▪ Caused by mutations of genes in the alpha Reversible oxidation and beta chains which results in the A form of hemoglobin that contains iron in the ferric (Fe3+) production of methemoglobin state Methemoglobinemia could be ACQUIRED or CONGENITAL (hereditary) o Ferric (Fe3+) – OXIDIZED FORM Treatment of acquired methemoglobinemia: o Ferrous (Fe2+) – REDUCED FORM o Removal of offending substance Also called Hemiglobin (Hi) ▪ Ex. Oxidant from drugs can be removed Formed within the erythrocytes in small amounts due to o Administration of ascorbic acid or methylene blue spontaneous oxidation (resulting from overload of oxidant o Use of oxygen inhalation techniques stress, owing to the ingestion of a strong oxidant drugs DIAGNOSIS o Oxidized iron: cannot bind oxygen Prevented from accumulating within the RBCs by reducing Peripheral blood film (presence of Heinz bodies or denatured oxidized heme by several enzyme systems (Methemoglobin hemoglobin) Reductase Pathway) Diaphorase enzyme screening test o NADH-methemoglobin reductase (diaphorase) is o To know whether the enzyme is deficient or not present in erythrocytes at a level sufficient to Methemoglobin quantitation counter methemoglobin o Levels can be detected by spectrum absorption o Maintains the normal concentration of instruments methemoglobin within RBCs: less than 1% o Peaks in the range of 620 to 640 nm at a pH of 7.1 ▪ Because oxidants can be taken through HEINZ BODIES some drugs or can be available in the Denatured hemoglobin environment, Diaphorase maintains a o Inclusions found in the cytoplasm normal concentration of methemoglobin o Considered as abnormal, thus phagocytosed by within the RBCs. macrophages o Methemoglobin Reductase - an enzyme that allows o Decrease RBC survival the ferric form (Fe3+) of Iron to be reduced back to the ferrous form (Fe2+) CHARACTERISTICS Has a brownish to bluish color, does not revert to red on exposure to oxygen When one or more iron atoms have been oxidized, hemoglobin molecule changes Figure 1. Heinz Bodies DMC COLLEGE FOUNDATION, INC. 1  [TRANS] HEMATOLOGY I: KATE CARLA RETES, RMT SULFHEMOGLOBIN ▪ Exogenous sources: exhaust of Chemically modified hemoglobin formed by irreversible automobiles and from industrial pollutants oxidation of hemoglobin by certain drugs such as: such as coal, gas, charcoal burning, and o Sulfonamides tobacco smoke o Phenacetin o COHb levels among smokers are as high as 15% o Acetanilide As a compensatory mechanism for possible hypoxia, these o Phenazopyridine individuals often express high hematocrit levels and Iron is in the ferrous form but its affinity for oxygen is lower polycythemia (Rodaks) than normal hemoglobin Toxic effects: headaches, dizziness, coma, convulsions In vitro: formed by the addition of hydrogen sulfide to Hb o 20 – 30% COHb - dizziness, headaches, and has a greenish pigment disorientation. o > 40% COHb - coma, seizure, hypotension, cardiac CHARACTERISTICS arrhythmias, pulmonary edema, and death Ineffective for oxygen transport (100x less affinity for Gives blood a cherry-red color, which is also imparted to the oxygen) skin of carbon monoxide poisoning patients Cannot be converted to normal hemoglobin Treatment: administration of hyperbaric oxygen Persists for the life of the cell (until the RBCs are removed LABORATORY SCREENING TESTS from the circulation) 1. Hemolyzing 0.5 mL of whole blood with distilled water Peaks at 620 nm on a spectral absorption instrument 2. Adding 1 mL of NaOH, 1.0 mol/L CARBOXYHEMOGLOBIN 3. More than 20% carboxyhemoglobin – appearance of Also called Carbihemoglobin or COHb sample is light cherry-red color The oxygen molecules bound to heme are replaced with Normal blood – mixture will turn brown carbon monoxide (CO) HEMOGLOBIN MEASUREMENT o So instead of oxygen molecule to attach to the Reference Method: CYANMETHEMOGLOBIN heme, carbon monoxide (CO) will attach to the heme Measures all types of hemoglobin except: SUFHEMOGLOBIN Caused by binding of carbon monoxide to heme iron Lysing agent is used to free hemoglobin from the red blood cells Hemoglobin has about 200 -240 times more affinity to carbon monoxide compared to oxygen Hb combines with potassium ferricyanide o In that sense, example, when you are exposed to Methemoglobin combines with potassium cyanide to form carbon monoxide, although hemoglobin can the stable pigment cyanmethemoglobin normally attach to oxygen, when there is exposure Used to quantify hemoglobin by reading the color change is to carbon monoxide (CO) the affinity is greater spectrophotometrically at 540 nm compared to the oxygen When RBCs are already lysed, the free hemoglobin which has o Carbon monoxide combines with hemoglobin ferrous forms of iron will be mixed with potassium slower than oxygen but union is much firmer ferricyanide o Although it binds slow, oce it is united, it is firmer It will become methemoglobin; it will be mixed with another and cannot be easily detached compound called potassium cyanide o Release of carbon monoxide is 10,000 times slower And the methemoglobin will become cyanmethemoglobin, than the release of oxygen which is the final product that will be measured at 540 nm in o Once the carbon monoxide is attached to the heme the spectrophotometer instrument iron, it will be harder for the body to release carbon IRON METABOLISM monoxide from the system IRON Termed as the “silent killer”, odorless and colorless gas - patients quickly become hypoxic Primary function of iron: oxygen transport May be detected by spectral absorption instrument at 541 Iron is more stable in the ferric state nm (540 nm according to Rodak’s) Most of the iron is in the ferric state (bound to transferrin, May be endogenous or exogenous ferritin) o Endogenous - inside or internal factors; > 2% of the Reduction of iron from the ferric to ferrous state requires total hemoglobin (Rodaks, 2016) NADPH and a number of enzymatic reactions o Exogenous - from outside factors or environment Most functional iron in humans is in the form of hemoglobin and myoglobin DMC COLLEGE FOUNDATION, INC. 2  [TRANS] HEMATOLOGY I: KATE CARLA RETES, RMT In Hemoglobin - ferrous iron is incorporated into NON-HEME IRON protoporphyrin IX (a ring of carbon, hydrogen, and nitrogen; o Leafy vegetables a heme precursor) o Legumes ¼ of iron is in storage form o Beans Non-heme iron is stored as FERRITIN OR HEMOSIDERIN in o Cereals hepatocytes of macrophages o Milk Small amount is bound to transferrin (protein transporter of SOURCES OF IRON iron) 1. Amount and type of iron from food FORMS OF IRON o Amount, meaning quantity of food intake that is rich Carrier of electrons in iron, and type, meaning whether it is rich in heme A catalyst for oxygenation, hydroxylation, and other crucial or non-heme iron. metabolic processes – due to the ability to cycle reversibly 2. Functional state of the gastrointestinal mucosa between ferrous and ferric states o It depends on how functional the GI mucosa is to Must be regulated carefully in its free forms - it plays a key absorb iron role in the formation of harmful oxygen radicals that can 3. Current iron stores damage cellular structures (e.g. hemoglobin of RBCs) o Storage of iron is primarily ferritin, so it depends on Iron exists as a free cation transiently them. Mostly bound or incorporated into various proteins 4. Erythropoietic needs o If we need to have more RBCs, then we also need IRON INTAKE AND LOSS SHOULD BE BALANCED more iron. Iron deficiency: occurs from inadequate intake or excessive IRON ABSORPTION AND EXCRETION loss through bleeding o Red blood cell functions start to decline (decrease in Maximal absorption happens in the duodenum and upper hemoglobin) jejunum. Iron overload: increased absorption or repeated blood transfusions o May cause iron toxicity and may produce organ damage and death IRON STATUS – depends on iron intake, iron bioavailability, and iron losses DIETARY IRON Bioavailability of iron depends on its chemical form in the food, whether inorganic or organic, or whether it is non-heme or heme, and the presence of other food items that promote or inhibit absorption. Absorbed in two forms: heme iron and non-heme iron Figure 2. Gastrointestinal tract 1. Heme iron (Fe2+): iron that is incorporated in the For transport of oxygen in Hb, iron must be in the ferrous hemoglobin molecule (absorbed more effectively) form (Fe2+) o The one with ferrous form To be absorbed, iron from food must be in the heme iron o Present in forms of hemoglobin, myoglobin, and (ferrous state/ Fe2+) or converted from the ferric non-heme heme enzymes iron to the soluble ferrous iron 2. Nonheme iron (Fe 3+): inorganic iron Conversion of ferric non-heme iron to the soluble ferrous o The one with ferric form form by DCYTB (duodenum-specific cytochrome b-like o Usually bound to transferrin, ferritin protein) Sometimes visible to developing erythroid cells Heme iron binds to the enterocyte (intestinal absorptive (Pappenheimer Bodies) cells) in the mucosal epithelium and is internalized SOURCES OF IRON HEME IRON Ferric is being converted to ferrous by DCYTB, and with the help of DMT o Liver (divalent metal transporter), ferrous goes back inside of the enterocyte to o Meat be internalized o Poultry o Fish DMC COLLEGE FOUNDATION, INC. 3  [TRANS] HEMATOLOGY I: KATE CARLA RETES, RMT Ferrous iron is transported across the duodenal epithelium by the apical divalent metal transporter-1 (DMT1) Ferrous iron is carried to the basolateral membrane (base and sides of the membrane) and exported to the portal circulation and mediated by FERROPORTIN (a basolateral transport protein) Ferroportin works in conjunction with HEPHAESTIN: a copper-containing iron oxidase (facilitates reoxidation of ferrous to ferric iron) Oxidized iron must be bound to transferrin to be transported Figure 4 Hemosiderin in the circulation o Some absorbed iron remains in the enterocyte as HEPCIDIN FERRITIN (stored form of iron) An antimicrobial peptide produced by the liver Acts as negative regulator of intestinal iron absorption FERRITIN VS HEMOSIDERIN Binds to the ferroportin receptor causing degradation of Ferritin and hemosiderin stores are found in the liver, bone ferroportin marrow, and spleen, with most in the liver. o Hepcidin is a regulatory peptide (helps regulate iron absorption) FERRITIN Results in trapping of iron in the intestinal cells main storage form of iron Hepcidin is: Component released by the reticuloendothelial system 1. An acute-phase reactant Labile form of iron storage, meaning that iron can get in and 2. A hormone produced by the hepatocytes to regulate out of this form quickly body iron levels o The form of our iron in ferritin is ferric, so it can be ✓ Absorption of iron in the intestine ferrous later or go back again to its original form. ✓ Release of iron in the macrophages HEPCIDIN PRODUCTION is directly proportional to BODY IRON LEVELS (↑ iron = ↑ hepcidin; vice versa) Figure 5. Actions of hepcidin Hepcidin reduces iron absorption in the enterocyte and Figure 3. Ferritin increases iron accumulation in the macrophage o It's called the storage form, because it can also be used in times of iron deficiency in the body. In those SOURCES OF IRON instances, the ferric will be reduced to ferrous in Dietary iron order for the body to use it. Iron recycled from extravascular hemolysis acute phase protein, which means that it goes up (increase) in certain conditions, like chronic inflammation. HEMOSIDERIN consists of ferritin and cell debris Stable form of storage Iron in this form is less readily accessible Cannot be used, because it cannot revert the ferric to ferrous form (heme form of iron) DMC COLLEGE FOUNDATION, INC. 4  [TRANS] HEMATOLOGY I: KATE CARLA RETES, RMT SUMMARY OF IRON ABSORPTION In the bone marrow: 1. Transferrin binds to a transferrin receptor on the cell membrane 2. Transferrin receptors are bound to erythroid progenitors and precursors that are rapidly dividing, except mature RBCs 3. Transferrin is taken into the cell by endocytosis 4. Endosome formation 5. Release of iron from transferrin by acidification of the endosome to pH 5.5 6. Iron transported across the endosomal membrane by DMT1 Humans: regulate iron by controlling absorption o Normal absorption of iron: 1 to 2 mg/day o Decreased iron stores: 3 to 4 mg/day ▪ If you have less iron, the body will absorb a larger amount of iron than its normal Figure 6. Summary of Iron Absorption absorption amount o Iron overload: 0.5 mg/day ▪ If you have more iron, the body will absorb Heme Iron Ferrous Form can be readily absorbed by the body a lesser amount of iron than its normal but the Non-Heme Iron Ferric Form must be converted to absorption amount Ferrous Form in order to be absorbed by the body o DCYTB (Duodenal Cytochrome b) is the one that converts the Ferric Form to Ferrous Form FERROKINETICS In order for the Ferrous form to go inside the enterocyte it Movement of Iron in the body starting from absorption will be facilitated by the DMT 1 (Divalent Metal Transporter) Includes the following: With the help of Ferroportin in conjunction with Hephaestin, o Transferrin Ferrous Form will become Ferric Form to be transported in o Transferrin receptor the Hepatic Circulation carried by the Transferrin o Ferritin Ferric Form will be carried by the transferrin to be delivered in the bone marrow TRANSFERRIN Hepcidin is present in order to regulate the Produced by hepatocytes absorption/release of iron in the enterocyte Major function is to transport iron (Fe3+) from the plasma to Additional Notes from Rodak’s the normoblasts in the marrow Iron can be absorbed in the intestines as heme from animal Transferrin binds to transferrin receptors on the normoblast food sources or as ionic iron, mostly from vegetable sources. membrane Once heme enters enterocyte by receptor-mediated Half-life: 8 days endocytosis, the iron is freed from protoporphyrin by heme oxygenase. Ferroportin is the only known protein that exports iron across TRANSFERRIN RECEPTORS cell membranes. Transferrin receptor: bind two molecules of transferrin When the body has sufficient stores of iron, the hepatocytes Transferrin to transferrin receptor affinity: depends on the can sense that and will increase the production of hepcidin, iron content and pH a protein that can bind to ferroportin leading to inactivation. Highest affinity: diferric transferrin and pH 7.4 It will result in a decrease in iron absorption. When iron starts to drop, the liver decreases hepcidin FERRITIN production and makes ferroportin active again. It will be able Apoferritin: protein component of ferritin without IRON to transport iron into the blood. Transferrin Receptors can bind to Ferric iron o When the pH of transferrin is equal to 7.4 IRON CYCLE AND TRANSPORT o What triggers for the ferric iron to be attached to Transferrin with iron – transferrin carries iron to transferrin? It is when the pH is 7.4 hematopoietic tissues which is in the bone marrow DMC COLLEGE FOUNDATION, INC. 5  [TRANS] HEMATOLOGY I: KATE CARLA RETES, RMT Diferric Transferrin: Transferrin with 2 Ferric Irons, is RBC DISTRIBUTION absorbed by the cell through endocytosis where it will form NORMAL DISTRIBUTION endosomes Endosomes acidify to pH 5.0/5.5 and it will trigger the Even distribution of erythrocytes in the thin portion release of Ferric Iron from the transferrin adjacent to the feather end of the blood smear When the transferrin has already detached with the ferric Red blood cells should be slightly separated from one iron, that transferrin will be called “Apo-transferrin” another or barely touching without overlapping In a blood smear, this area is called “ZONE OF LABORATORY ASSESSMENT FOR IRON STORES MORPHOLOGY” (CRITICAL AREA OF READING/MONOLAYER AREA) Serum iron concentration: refers to the Fe3+ bound to transferrin PARTS OF A PERIPHERAL BLOOD SMEAR o Exhibits diurnal variation – highest in the morning and lowest in the evening o Decreased in iron-deficiency anemia and inflammatory disorders TIBC (Total iron-binding capacity): total number of available sites of transferrin % Transferrin saturation: percentage of transferrin carrying iron Serum ferritin: levels of stored iron; declines early in iron- deficiency anemia Free Erythrocyte Protoporphyrin – may be seen in excess when there is IDA Figure 8. Parts of Blood Smear Prussian blue staining (Iron staining) – test to see iron concentration in macrophages, nucleated RBCs and Label: Name of px, DOB, time and date of collection, initials reticulocytes of phlebotomist o Sideroblasts – Immature RBCs,either nucleated or Application point: where blood is dropped reticulocytes, that has iron content with it THICK AREA o Siderocytes – Mature RBCs that has iron content in o RBCs may overlap, making them unsuitable for it evaluation THIN END OF THE BLOOD SMEAR o RBC distribution is irregular with artifactual shapes and size distortions Figure 7. Smear with Erythroids This particular smear has been stained with Iron Staining/Prussian Blue. Cells pointed by the arrows are erythroblasts/immature RBCs with iron (blue colored stains) Figure 9. Thick area of blood smear ABNORMAL RED BLOOD CELL Normal: biconcave disc of 6-8 um diameter Has a central pallor (⅓ of diameter) on smear No inclusions Figure 10. Thin area (feathered edge) of blood smear DMC COLLEGE FOUNDATION, INC. 6  [TRANS] HEMATOLOGY I: KATE CARLA RETES, RMT ABNORMAL DISTRIBUTION ROULEAUX FORMATION RBCs are not separated from one another Appear in short or long stacks (rouleaux) resembling “stack of coins” Four or more cells make up each formation, leaving much of the field empty of cells (increased white space) Arrangement of cells with their biconcave surfaces close together Figure 14. Artifactual CLINICAL SIGNIFICANCE: AUTOAGGLUTINATION Characteristic of hyperproteinemia and multiple myeloma due to increased amount of globulin (protein) Occurs as RBCs aggregate (rather than stacked like coins) Confirmed with erythrocyte sedimentation rate (ESR) into random clusters or masses when exposed to antibodies in the plasma Found when fibrinogen is increased (infections, pregnancy) Outline of individual cell is not seen May be classified as: TRUE ROULEAUX or ARTIFACTUAL Occurs when an individual’s RBCs agglutinate in his/her own plasma or serum More likely to be observed in connection with certain hemolytic anemias, infections (increase level of antibodies which makes RBCs prone to agglutination) Example: Cold Agglutinin Disease o RBCs clump on a blood film at cold temperatures Figure 11. Rouleaux Formation Figure 15. Autoagglutination Figure 12. Rouleaux Formation Mechanism RBC MORPHOLOGY IN-VIVO Rouleaux are stacks of erythrocytes. Their formation is promoted by acute phase reactant and immunoglobulin Non-nucleated, biconcave disc (discocyte) and hampered by albumin. The degree of rouleaux Shape is suited for RBCs task of gas transport and survival in formation is one of the major determinants of ESR the circulation (Erythrocyte Sedimentation Rate) IN-VITRO (BLOOD SMEAR) Flattened and thus has a round appearance with a central pallor (⅓ of the diameter of the cell) Diameter: 6-8 um Uniform in size, shape and hemoglobin concentration No inclusions in normal red blood cell Approximately the same size or slightly smaller than the nucleus of a small lymphocyte Figure 13. True Rouleaux DMC COLLEGE FOUNDATION, INC. 7  [TRANS] HEMATOLOGY I: KATE CARLA RETES, RMT MACROCYTIC RED BLOOD CELLS o Megaloblastic anemia (>100 fL) MICROCYTIC RED BLOOD CELLS o Iron deficiency anemia/ Iron overload conditions o Thalassemia o Anemia of chronic disease o Sideroblastic anemia Figure 16. Central Pallor Figure 18. Anisocytosis Figure 17. Small lymphocytes MACROCYTIC RED BLOOD CELLS Small lymphocytes: can be mistaken as nucleated RBCs Size: > 8 um MCV: > 100 fL COMMON CAUSES: TERMINOLOGIES o Vitamin B12 or Vitamin B9 (Folate) deficiency ANISOCYTOSIS o Alcoholism with or without hepatic disease Variation in size o Stem cell disorders (aplastic anemia) Normal, non-nucleated RBCs that have just left the bone POIKILOCYTOSIS marrow sinusoids are slightly macrocytic and appear in Variation in shape stained peripheral blood films POLYCHROMASIA Prematurely released (“shift cells”), occur as a result of stimulated erythropoiesis Variation in color MCV (MEAN CORPUSCULAR/CELL VOLUME) Indicates average size of erythrocytes o MCV: 80-100 Fl ▪ RBCs (NORMOCYTIC) – normal size, even if it demonstrates a minor population of smaller or larger cells o Decreased (< 80 fL) in microcytic anemias: ▪ Iron Deficiency Anemia, Thalassemia, Sideroblastic anemia o Increased (> 100 fL) in macrocytic anemias: ▪ Megaloblastic anemia Figure 19. Macrocytic RBC ANISOCYTOSIS OVAL MACROCYTES Variation in red cell population size Normal RBC: 6-8 um Markedly increased MCV: >125 fL MCV: 80-100 fL Megaloblastic erythropoiesis: caused by VITAMIN B12 OR VITAMIN B9 (FOLATE) DEFICIENCY Anisocytosis: Hemoglobin content increases as the cell size increases in o Microcytes: < 6 um size o Macrocytes: > 8 um Macrocyte: without a central pale area DMC COLLEGE FOUNDATION, INC. 8  [TRANS] HEMATOLOGY I: KATE CARLA RETES, RMT To the left: older and smaller; fragmented If RDW increases: causes variation Figure 20. Oval Monocyte MICROCYTIC RED BLOOD CELLS Figure 23. Microcytic Anemia Small erythrocytes with reduced size; normal or decreased hemoglobin Hemoglobin concentration becomes depressed and RBCs become more microcytic and hypochromic MCV: < 80 fL Causes: o Anemia of chronic disease o Thalassemia (defective globin synthesis) o Iron – deficiency anemia o Sideroblastic anemia (defective protoporphyrin Figure 24. Macrocytic Anemia synthesis) o Spherocytosis (small, but lack central pallor) RBC COLOR POLYCHROMASIA Occurs subsequent to excessive production of red blood cell precursors in response to anemic stress Polychromasia is observed in the following: o When the bone marrow is responding to anemia o When therapy is instituted for iron deficiency anemia or megaloblastic anemia o When the bone marrow is being stimulated as a result of a chronic hematologic condition, such as thalassemia or sickle cell disorders Figure 21. Microcytic RBC RDW (RBC DISTRIBUTION WIDTH) Refers to the homogeneity of the RBC population size A large RDW – wide variation in the RBC diameter Figure 25. Polychromasia HYPOCHROMIA Hypochromic RBCs: less in color o Iron deficiency anemia/Iron overload conditions o Thalassemia o Anemia of chronic disease o Sideroblastic anemia Figure 22. RDW To the right: younger and bigger DMC COLLEGE FOUNDATION, INC. 9  [TRANS] HEMATOLOGY I: KATE CARLA RETES, RMT Figure 26. Hypochromia Rich in color; no central pallor; RBCs are very much filled with hemoglobin RBC SHAPE NORMAL SHAPE shows little or no shape variation Variation on the edges of films: artifact of preparation Recognition of various shapes is helpful in differentiation of anemias Sometimes caused by: o structural or biochemical changes o metabolic state in the cell o hemoglobin molecule abnormalities ABNORMAL SHAPE / POIKILOCYTOSIS Variation in the shape of red blood cell Presents visual clues for what might be the source of the patient’s hematologic problems Caused by decreased red blood cell destruction, increased destruction, or defective splenic function 3 categories: o Membrane Abnormalities o Secondary to Trauma o Abnormal Hemoglobin Content Commonly associated disease states: o Severe anemia o Certain shapes helpful diagnostically DMC COLLEGE FOUNDATION, INC. 10