Hematology 1

Week 2 Terence L. Eday, RMT, MT(ASCPi), MPH

Hematology 1 Week 2

THE ERYTHROCYTE

Introduction
One of the first microscopic elements recognized and described after the discovery of the microscope 1817, Francois Magendie erroneously surmised RBCs as air bubbles 1865, Felix Hoppe-Seyler discovered the hemoglobin

The Erythrocyte

ERYTHROPOIESIS AND RBC MATURATION

Erythropoiesis and RBC Maturation

Erythron refers to the totality of all stages of erythrocytes Erythropoiesis production of erythrocytes; normally an orderly process through which the peripheral concentration of erythrocytes is maintained at a steady state 120 days 10 days circulating life span for mature cells

Erythroid Progenitor Cells

Committed (unipotential) erythroid progenitor cell compartment: 1) burst-forming unit-erythroid (BFU-E) and 2) colony-forming unit-erythroid (CFU-E) BFU-Es progenitor cells that give rise to a burst or multifocal colony of cells in vitro assay in 10 to 14 days CFU-Es intermediate precursor of the earliest morphologically recognizable erythroid precursor, the pronormoblast

Erythroid-Maturing Cells
Erythroblasts collective term for nucleated erythrocyte precursors in the BM Normoblasts maturation sequence is normal Reticulocytes young erythrocytes with residual RNA but without a nucleus Two terminology systems: 1) normoblast, and 2) rubriblast

Erythroid-Maturing Cells
Stages (from most immature to mature): pronormoblast (rubriblast), basophilic normoblast (prorubricyte), polychromatophilic normoblast (rubricyte), orthochromatic normoblast (metarubricyte), reticulocyte, and erythrocyte

Pronormoblast (Rubriblast)

Basophilic normoblast (Prorubricyte)

Polychromatophilic normoblast (rubricyte)

Orthochromatic normoblast (metarubricyte)

Reticulocyte

Polychromatophilic RBC (Diffuse basophilia)

Mature Erythrocyte

Erythroblastic Islands (EI)

It is where erythropoiesis occurs Consists of concentric circles of developing erythroblasts clustered around a central macrophage Erythroblasts adhere to central macrophage by variety of cytoadhesion molecules [i.e. fibronectin (Fn) and thrombospondin (Tsp)] Surrounding the EI are fibroblasts, macrophages, and endothelial cells

The Erythrocyte

ERYTHROCYTE MEMBRANE

Erythrocyte Membrane
Steven Hedin demonstrated the osmotic properties and selective permeability of the RBC Karl Landsteiner observed that human sera caused clumping of the RBC; divided individuals into three groups A, B, and C Essential for development and function In association with the membrane skeleton, provided the dual characteristics of strength and flexibility

Erythrocyte Membrane Composition

Erythrocyte Membrane Composition
Lipids and glycolipids (~45%) Unesterified cholesterol Phospholipids Phosphatidylinositol (PI) Phosphatidylethanolamine (PE) Phosphatidylserine (PS) Phosphatidylcholine (PC) / Lecithin Sphingomyelin (SM) Lycophospholipids (Lycophosphatidylycholine, Lycophosphatidylethanolamine) Glycolipids

Erythrocyte Membrane Composition

Erythrocyte Membrane Composition
Proteins and glycoproteins (~55%) Integral proteins Glycophorins A, B, C, D E (carry antigens on exterior of membrane) Band 3 (attaches skeletal lattice to membrane lipid bilayer, anion exchange channel) Peripheral proteins (form membrane skeletal lattice and attach it to membrane) Spectrin ( and polypeptides) Actin (band 5) Ankyrin (band 2.1) Band 4.2 Band 4.1 Adducin (band 2.9) Band 4.9 (dematin) Tropomyosin (band 7) Tropomodulin (band 5)

RBC Membrane

Membrane Permeability
Concentration of Cations in the Erythrocyte versus Plasma Cation Erythrocyte Plasma (mmol/L) (mmol/L)
Sodium (Na+) Potassium (K+) Calcium (Ca++) Magnesium (Mg++) 5.4-7.0 98-106 0.0059-0.019 3.06 135-145 3.6-5.0 2.1-2.6 0.65-1.05

The Erythrocyte

ERYTHROCYTE METABOLISM

Erythrocyte Metabolism
Role of Metabolic Pathways in Erythrocyte
Metabolic Pathways Glycolytic pathway Key Enzymes Function Hemapathology Hemolytic anemia Hereditary PK deficiency Hemolytic anemia Hereditary G6PD deficiency Glutathione reductase deficiency Phosphofructokinase Produces ATP Pyruvate kinase accounting for 90% of glucose consumption in RBC Glutathione Provides NADPH and reductase glutathione to Glucose-6-phosphate reduce oxidants dehydrogenase that would shift (G6PD) the balance of oxyhemoglobin to methemoglobin

Hexose monophosphate shunt

Erythrocyte Metabolism
Role of Metabolic Pathways in Erythrocyte
Metabolic Pathways Rapoport-Leubering Key Enzymes BPG-synthase Function Controls the amount of 2,3-BPG produced, which in turn affects oxygen affinity of hemoglobin Hemapathology Hypoxia

Methemoglobin reductase

Methemoglobin reductase

Protects hemoglobin Hemolytic anemia from oxidation via Hypoxia NADH (from glycolytic pathway) and methemoglobin reductase

The Erythrocyte

ERYTHROCYTE KINETICS

Introduction
Dr. Dennis Jourdanet, a French physician, noted that patients with altitude sickness exhibited symptoms similar to patients with anemia at sea level

Erythrocyte Concentration
Normal concentration varies with sex, age, and geographic location Males have higher erythrocyte concentration than females after the age of puberty due to testosterone

Individuals living at high altitudes have higher mean of erythrocyte concentration

Regulation of Erythrocyte Production

Erythropoiesis is triggered by erythropoietin (EPO) Erythropoiesis is influenced by cytokines (SCF, IL-3, GM-CSF, and EPO) EPO principal cytokine essential for terminal erythrocyte maturation

Erythropoietin (EPO)
Thermostable renal glycoprotein hormone MW ~34,000 daltons Secreted by renal cortical interstitial cells Active at ~130,000 IU/mg of protein Normal plasma: 5 25 IU/L

The Erythrocyte

ERYTHROCYTE DESTRUCTION

Erythrocyte Catabolism
Life span:
Normal adult: 120 days 10 days Pre-mature infant: 35 to 50 days Fetus: 60 to 70 days

As the it ages:
1. Membrane becomes less flexible 2. Concentration of cellular hemoglobin increases 3. Enzyme activity, particularly glycolysis diminishes

Extravascular Destruction

Intravascular Destruction

The Hemoglobin
Hematology 1 Week 2

Introduction
1862, Felix Seyler identified hemoglobin Hemoglobin (Hb) occupies ~33% of the erythrocyte volume; 90% of the cells dry weight Each RBC contains between 28 to 34 pg of hemoglobin Most hemoglobin synthesis occurs at the polychromatophilic normoblast

Introduction
Normal hemoglobin concentration in adult is ~15 g/dL with a total blood volume of 5000 mL Total body mass of hemoglobin: 750 g 6.25 g/day = amount of Hb lost and synthesized each day 2 x 1011 cells/day

Hemoglobin

HEMOGLOBIN STRUCTURE

Hemoglobin Structure

Normal Types of Hemoglobin According to Developmental Stage

Developmental Stage Type Gower I Gower II Portland Globin Chains

Embryonic

22 22 22 22 22 22 22 22 22 22 22 22

Fetal

HbF HbA HbA2

>1 year old

HbF HbA HbA2

Adult

HbA HbA2 HbF

Hemoglobin

HEMOGLOBIN SYNTHESIS

Heme
Iron-chelated porphyrin ring that functions as a prosthetic group of a protein Porphyrin ring, protoporphyrin IX composed of a flat tetrapyrrole ring with ferrous iron (Fe++) inserted into the center

In deoxygenated state, the 6th electron pair is occupied by water

Heme Synthesis

Globin
Synthesis is directed by eight genetic loci Genes that encode the alpha globin chains are on chromosome 16 Non-alpha are encoded on chromosome 11 Synthesis of globin peptide chains occurs on polyribosomes in the cytoplasm of developing erythroblasts

Hemoglobin

REGULATION OF HEMOGLOBIN SYNTHESIS

Regulation of Hemoglobin Synthesis

Mechanisms:
Activity of the erythroid enzyme 5aminolevulinate synthase Activity of phorphobilinogen deaminase (PBGD) Concentration of iron Regulation of globin chain synthesis

Iron regulatory protein (IRP) primary physiologic iron sensor; previously referred to as iron responsive element-binding protein (IRE-BP)

Hemoglobin

HEMOGLOBIN FUNCTION

Oxygen Transport
Oxyhemoglobin hemoglobin with bound oxygen Deoxyhemoglobin hemoglobin without oxygen Oxygen affinity the ease with which hemoglobin binds and releases oxygen

Oxygen Dissociation Curve (ODC)

Factors That Affect Oxygen-Hemoglobin Affinity

Increase Affinity
Increased oxygen Decreased carbon dioxide Decreased pH Decreased temperature Decreased 2,3-BPG

Decrease Affinity
Increased carbon dioxide Increased hydrogen ions / pH Increased Temperature Increased 2,3-BPG

Hemoglobin

ACQUIRED NONFUNCTIONAL HEMOGLOBINS

Methemoglobin
Hemoglobin with iron in the ferric (Fe+++) state Incapable of combining with oxygen Decreases the oxygen-carrying capacity of blooo Increase oxygen affinity of the remaining normal hemoglobin Reduction of methemoglobin is carried out by NADH methemoglobin reductase

Methemoglobin
Increased levels are formed when exposed to oxidizing chemicals or drugs Color crayons containing aniline can cause methemoglobinemia if ingested Can be reduced by medical treatment with methylene blue or ascorbic acid Blood sample can be chocolate brown in color when compared to a normal blood specimen

Sulfhemoglobin
Stable compound formed when a sulfur atom combines with the heme group of hemoglobin Binds to oxygen with an affinity only onehundredth that of normal hemoglobin Produces bright green pigment Associated with occupational exposure to sulfur compounds, polluted air, and certain drugs

Sulfhemoglobin
Formed on exposure of blood to trinitrotoluene, acetanilide, phenacetin, and sulfonamides Elevated in severe constipation and in bacteremia with Clostridium welchii Isoelectric focusing confirmatory test Not measured by cyanmethemoglobin method

Carboxyhemoglobin
Formed when hemoglobin is exposed to carbon monoxide Incapable of transporting oxygen High level impart a cherry red color to the blood and skin High levels CoHb with high levels of deoxyhemoglobin can give a purple pink color