Hematology - 03 Erythrocytes

HEMATOLOGY ERYTHROCYTES OUTLINE • Erythrocytes o Erythrocytes o RBC Structure o RBC Metabolism o RBC Membrane • RBC Anomalies o Anisocytosis o Anisochromia o Polychromasia o Poikilocytosis o RBC Inclusion o RBC Morphology Grading Chart o Abnormal Erythrocyte Distribution ERYTHROCYTES ERYTHROCYTES • aka Red Blood Cells (RBC) • Anucleated, biconcave disc shaped cells • Average Life Span: 120 days o Due to absence of nucleus • Reference values: o Male: 4.0 – 5.2 x 10 12 /L o Female: 3.6 – 5.6 x 10 12 /L o Newborn: 5.0 – 6.5 x 10 12 /L FUNCTIONS: • Oxygen transport • Removal of metabolic wastes • Has 2 main requirements to perform function o Energy source ▪ During expulsion of nucleus in orthochromic normoblast, other organelles are also expelled including mitochondria ▪ RBC has no mitochondria (important for energy production) ▪ RBC will rely on external glucose source for energy production ▪ Energy is generated through anaerobic glycolysis o Intact structure (RBC membrane) RBC STRUCTURE • Anucleated • Size : 6-8 um • Thickness : 1.5-2.5 um o 1.5 - central pallor; 2.5 - thickest portion • Shape : Biconvace disc • Cytoplasm : Salmon pink with central pallor area (1/3 of RBC diameter) • Average volume : 80-100 fL (90 fL) • Surface area : 140 um 2 • Increased surface-to-volume ratio (allow the deformability of the cell) RBC METABOLISM • Four Pathways : 1. Embden-Meyerhof Pathway 2. Hexose Monophosphate Shunt 3. Methemoglobin Reductase Pathway 4. Rapoport-Luebering Pathway ENERGY-DEPENDENT RBC METABOLISM • Energy is required to fulfill RBC processes • RBC processes dependent on energy o Intracellular cationic gradient maintenance o Maintenance of membrane phospholipid distribution o Maintenance of skeletal protein deformability o Maintenance of functional hemoglobin with ferrous iron (methemoglobin reductase pathway) o Protecting cell proteins from oxidative denaturation (hexose monophosphate shunt - HMS) o Glycolysis initiation and maintenance o Glutathione synthesis (from HMS) o Nucleotide salvage reactions EMBDEN-MEYERHOF PATHWAY • Anaerobic pathway of glucose metabolism o Does not consume oxygen during the process • Source of 90-95% of RBC requirement • Requires glucose to produce ATP (byproduct) • Product : o Lactate o Pyruvate o 2 ATP (byproduct) - high energy phosphate source; high reservoir for energy

HEXOSE-MONOPHOSPHATE SHUNT • aka Pentose Phosphate Pathway, phosphogluconate pathway • Aerobic and oxidative pathway • Functionally dependent on Glucose-6-phosphate dehydrogenase pathway (G-6-PD) • Detoxifies accumulated peroxides (oxidants that oxidizes heme iron proteins and lipids) o To prevent oxidation of heme iron (Fe 2+ → Fe 3+ ) ▪ Fe 3+ has no capacity to bind oxygen • Produces reduced glutathione o Prevents oxidative denaturation of hemoglobin • When glucose 6-phosphate is converted to 6-phospho-gluconate to pentose phosphate by the action of G6PD enzyme, we reduce NADP to its reduced form (NADPH) • When NADPH is available, glutathione reductase is capable to reduce the oxidized form of glutathione to its reduced form • Reduced glutathione can prevent oxidative denaturation of Hgb • Oxidative denaturation may occur due to the presence of peroxide (H2O2) • Reduced glutathione is able to reduce peroxide to water and oxygen with the aid of glutathione peroxidase • Cannot reduce methemoglobin ferric iron to ferrous iron METHEMOGLOBIN-REDUCTASE PATHWAY • Maintains iron in the hemoglobin molecule in the Ferrous (Fe 2+ ) state by the action of Methemoglobin reductase (cytob5r). o Cytochrome b5 reductase - new term for Cytob5r • It is important to maintain the ferrous state because only ferrous state can bind oxygen • Iron in Hgb molecule is constantly exposed to oxygen which is an oxidizing agent leading to Ferric state • If Hgb molecule has ferric ion, it is already oxidized o Oxidizes ferric ion, it is now called methemoglobin • NADH/NADPH is important to maintain the pathway o Allow methemoglobin reductase to reduce methemoglobin to hemoglobin by reducing ferric iron to its ferrous state • MRP is more efficient in producing NADPH RAPOPORT-LUEBERING PATHWAY • Can be called, Rapaport-Luebering • Third metabolic shunt • Generates 2,3-bisphosphoglycerate (2,3-BPG, 2,3-DPG) • Regulates affinity of hemoglobin to oxygen o 2,3-BPG competes with oxygen for hemoglobin o When 2,3-BPG binds with Hgb, oxygen can no longer bind with the Hgb allowing oxygen to be released and delivered to the tissues HEMOGLOBIN AFFINITY TO OXYGEN Factors Increased affinity to O 2 Decrease affinity to O 2 pH ↑ (alkaline) ↓ (acidic) Partial pressure of CO 2 (amount of CO 2 in blood) ↓ ↑ 2,3-BPG concentration ↓ ↑ Temperature ↓ ↑ Presence of non-functional hemoglobin species Cannot bind oxygen OXYGEN DISSOCIATION CURVE • A curve produced when pO2 and hemoglobin affinity to oxygen are plotted on a graph • Shift to the left – increased hemoglobin affinity to oxygen o ↑ O 2 affinity o ↑ pH o ↓ pCO 2 o ↓ 2,3-BPG o ↓ Temperature • Shift to the right – decreased hemoglobin affinity to oxygen o ↓ O 2 affinity o ↓ pH o ↑ pCO 2 o ↑ 2,3-BPG o ↑ Temperature RBC MEMBRANE • Functions : 1. It gives the RBC its shape 2. It provides skeletal system for RBC 3. It maintains Osmotic balance 4. It facilitates transportation of gases

• RBC Membrane Deformability o The increased surface-to-volume ratio allows RBC to stretch undamaged 2.5 times their resting diameter. ▪ High SA = Low Vol. o Increased cytoplasmic viscosity compromised its deformability; High Hgb increase viscosity ▪ High Hgb in RBC transforms biconcave shape to spherical shape o RBC membrane lipid component maintains its strength and deformability o Allow RBC to fit into tiny blood vessels • Consist of : o Lipids (40%) o Proteins (52%) ▪ Integral Proteins (Transmembrane) ▪ Peripheral Proteins (Cytoskeletal/skeletal) o Carbohydrates (8%) RBC Membrane Lipids • Responsible for the semi-permeability of the cell • Lipid Bilayer : o Hydrophilic polar head group ▪ Directed toward the aqueous plasma and the cytoplasm ▪ Composed of phospholipids • Phosphatidylcholine • Sphingomyelin o Hydrophobic nonpolar tail ▪ Arranged to form the central layer of lipid bilayer ▪ Directed inside in the central part of the layer ▪ Composed of phospholipids • Phosphatidylserine • Phosphatidylethanolamine • Phosphatidylinositol • Cholesterol – confers tensile strength to the lipid bilayer. o Extremely hydrophobic lipid o Increased cholesterol in RBC membrane make membrane stronger but loses elasticity ▪ Looser elasticity decreases life span • Glycolipids – forms the glycocalyx with integral proteins o Sugar-containing lipids o Prevent microbial attacks o Protect RBC from mechanical damage o Form some of blood group antigens • Contain 1:1 ratio of phospholipid to cholesterol; equal portion of phospholipid and cholesterol o Important to the deformability of the RBC RBC MEMBRANE PROTEINS • Integral Proteins o Aka transmembranous proteins o Penetrates the lipid bilayer o Has high sialic acid residue (zeta potential) ▪ Zeta potential - repulsive electric current responsible why RBC do not attract one another ▪ Prevent RBC from touching each other o Functions : ▪ Transport site ▪ Adhesion site ▪ Signaling receptors o Supports carbohydrate-defined blood group antigen (glycoprotein blood group antigen)

o Band 3 - most abundant ▪ Function as anion transporter ▪ Facilitate chloride ion transport ▪ ABH antigens - present in ABO blood group o Glut-1 o PAS bands ▪ Glycophorins ▪ Also contain determinants for other blood groups • Peripheral Proteins o Aka cytoskeletal/skeletal proteins o Located adjacent to cytoplasmic membrane lipid layer o Provides lateral or horizontal membrane stability o Provide the membrane cytoskeleton of the cell o Spectrin (α, ß) – principal proteins (25-30%) ▪ Responsible for the elasticity of the cell (with actin and protein 4.1) ▪ Spectrin dimer - alpha & beta; connection between alpha & beta spectrin; provides elasticity to the cell o Actin (4.5%) RBC ANOMALIES 1. Variation in size 2. Variation in shape 3. Color variation 4. RBC inclusions 5. Alteration in RBC distribution on a peripheral blood smear ANISOCYTOSIS • Variation in size • Normal RBC: 6-8 um in diameter (normocyte) • Mean Cell Volume (MCV): 80-100 fL (90 fL) • Macrocyte : >8 um o MCV : >100 fL o Seen in : Pernicious anemia, Liver cirrhosis, Aplastic anemia o Megalocyte : MCV of >120 fL o Seen in : Pernicious anemia, Vitamin B12 and folic acid deficiency • Microcyte : <6 um o MCV : <80 fL o Seen in : IDA, Thalassemia, Hemolytic anemia, Sideroblastic anemia ▪ IDA - Iron-deficiency anemia • Ways to detect anisocytosis : 1. Using the nucleus of small lymphocyte in a peripheral blood ▪ Small lymphocyte - 7-10 um ▪ Nucleus of small lymphocyte is same size as RBC 2. Mean Cell Volume (MCV) value ▪ Normocyte - 80-100 fL ▪ Macro - >100fL ▪ Miro - <80fL 3. Red Cell Distribution Width (RDW) value ▪ Index of anisocytosis ▪ Coefficient of variation of RBC volume ▪ Reference value (CV): 11.5-14.5% ▪ Normal RDW - bell shape within 80-100 • 11.5-14.5% RDW ANISOCHROMIA • Variation in color • Reflects the hemoglobin content of RBC o Hgb provides pigment o Low Hgb - pale discoloration o High Hgb - strong red color • Normal RBC (Normochromia) : salmon-pink with central pallor area approx. 1/3 of the diameter. (central pallor has no Hgb) • Hypochromia o Central pallor area >1/3 of RBC diameter o Usually microcytic o Lower Hgb content • Hyperchromia o aka Spherocytes (latest term) ▪ if Hgb is higher than normal, it loses its biconcave shape becoming spherical in shape o Lacks central pallor area o Higher Hgb content Normochromic Hypochromic

Hyperchromic • Grading of Hypochromia Grading Central Pallor Area 1+ ½ of RBC diameter 2+ 2/3 of RBC diaemeter 3+ ¾ of RBC diameter 4+ Thin rim of hemoglobin • Parameters of Anisochromia o Central Pallor Area ▪ Normochromia - 1/3 ▪ Hypochrmia - >1/3 ▪ Hperchromia (sphero) - no central pallor o Mean Cell Hemoglobin (MCH) ▪ Average weight of hemoglobin in individual RBC ▪ Reference range (Normochromia): 26-32 pg • Hypochromia : <26 pg • Hyperchromia : >32 pg o Mean Cell Hemoglobin Concentration (MCHC) ▪ Average concentration of hemoglobin in each individual RBC ▪ Reference Range (Normochromia): 32-36 g/dL or % ▪ Hypochromia : <32 g/dL ▪ Hyperchromia : >36 g/dL (spherocytic) POLYCHROMASIA • RBC with different degrees of staining affinity • Slightly larger RBC with bluish tinge (Wright stain) o Bluish tinge is due to residual RNA • Indicates reticulocytosis o Reticulocytes use supravital stain ▪ Aka polychromatic erythrocyte (Wright stain) • Grading of Polychromasia : (per 100 RBC) Grading Percentage of Polychromatophilic RBCs Slight 1% 1+ 3% 2+ 5% 3+ 10% 4+ >11% POIKILOCYTOSIS • Variation is shape • Normal (Discocyte): round biconcave shape • Poikilocytes : o Spherocytes o Stomatocytes o Acanthocytes o Echinocytes o Burr cells o Ovalocytes o Dacryocytes o Knizocytes o Keratocytes o Schistocytes o Blister cells o Drepanocytes o Degmacyte o Leptocytes o Biscuit cells o Bronze elliptocytes o Semilunar bodies • Spherocytes o Spherical RBC o No central pallor area ▪ Due to increased Hgb concentration o Decreased surface-to-volume ratio o May be wrongfully reported ▪ Feathery edge - normally has spherocytes ▪ Only read in the perfect area (area where RBC are grossly touching each other but not overlapping) o Seen in: ▪ Hereditary spherocytosis ▪ Hemolytic anemia ▪ Burns (microspherocytes) • Stomatocytes o aka Mouth cells o Elongated RBCs with a slit-like central pallor (resemble mouth) o Seen in: ▪ Rh null Syndrome ▪ Alcoholism ▪ Hereditary Stomatocytosis ▪ Electrolyte imbalance ▪ Severe liver disease ▪ Thalassemia minor ▪ Glutathione deficiency ▪ Infectious mononucleosis • Acanthocytes o aka Thorn cells, Spur cells o RBC with irregular spiculated surface (spike-like) o Seen in: ▪ Abetalipoproteinemia ▪ Spur cell anemia ▪ McLeod Syndrome

▪ Liver cirrhosis ▪ Hemangioma ▪ Post-heparin administration ▪ Neonatal hepatitis ▪ Post-splenectomy (after surgical removal of spleen) • Echinocytes o RBCs with regularly spiculated surface ▪ Spikes - rounded tip o Seen in: ▪ Artifactual drying of blood smear ▪ Effects of salicylates or barbiturates ▪ Bile acid abnormalities ▪ Osmotic imbalance • Burr Cells o aka Sea-Urchin Cells o RBC with regularly spiculated surface ▪ Spike - pointed tips o Seen in: ▪ Uremia ▪ Acute Blood Loss ▪ Pyruvate Kinase deficiency ▪ Stomach Cancer • Ovalocytes o Oval-shaped RBC o Elliptocytes – rod-, sausage-, cigar-shaped RBC ▪ More elongated than ovalocytes o Seen in ▪ Hereditary Elliptocytosis (presence of both ovalocyte & elliptocyte) ▪ Hemoglobin C disease ▪ Hemolytic anemia ▪ IDA ▪ Pernicious anemia ▪ Sickle cell trait ▪ Thalassemia • Dacryocytes o aka Teardrop cells (daCRYocytes) o Pear-shaped or tear-shaped RBCs o Seen in: ▪ Primary myelofibrosis ▪ Pernicious anemia ▪ Homozygous Beta-thalassemia ▪ Hemolytic anemia ▪ Myelopthisic anemia ▪ Myeloid metaplasia • Knizocytes o Resembles a pinched bottle o Seen in: ▪ Hemolytic anemia ▪ Hereditary spherocytosis • Keratocytes o Partially deformed RBCs but not cut o Spiculated resembling two horns o Spindle-, half moon-shaped RBC o Seen in: ▪ Disseminated Intravascular Coagulation • Schistocytes o aka schizocytes, helmet cells o Fragmented RBCs o Remains of ruptured blister cells o Seen in: ▪ Prosthetic implants (artificial heart valve) ▪ Uremia ▪ Infantile pyknocytosis ▪ Disseminated Intravascular Coagulation ▪ Severe burns

▪ Microangiopathic hemolytic anemia ▪ Renal transplant rejection • Blister Cells o Erythrocyte containing one or more vacuoles that resemble skin blisters o Keratocytes and schistocytes are produced o Seen in: ▪ Pulmonary embolism in sickle cell anemia ▪ Microangiopathic hemolytic anemia • Drepanocytes o aka Meniscocyte, Sickle cell o Sickle-cell or crescent-shaped RBCs o Results from gelation of polymerized deoxygenated Hemoglobin S (abnormal form of Hgb) o Two forms: ▪ Oat-shape cells • Crescent-shaped RBC w/ less pronounced projection • If reoxygenated, RBC reverts back to biconcave shape ▪ Irreversible Sickle Cell (ISC) • Crescent-shaped RBC with long projection • If reoxygenated, the shape remans o Seen in: ▪ Sickle cell anemia • Common among African-American • Resistant to ▪ Hemoglobin SC disease • Degmacyte o aka Bite cells o RBC with one or more semicircular portions removed from the margin ▪ Different from keratocytes o Seen in oxidative-related hemolysis in G6PD deficiency • Leptocytes o aka Target cells, Mexican Hat Cells, Codocytes o RBC with centrally stained area with a thin outer rim of hemoglobin o Seen in: ▪ Thalassemia ▪ Lecithin-Cholesterol Acyltransferase (LCAt) deficiency ▪ Hepatic disorder ▪ Iron deficiency anemia ▪ Hemoglobin C disease ▪ Post-splenectomy • Biscuit Cells o Folded RBC o Seen in hemoglobin SC disease • Bronze Elliptocytes o Bipolar or central distribution of hemoglobin o Seen in Sickle cell anemia RBC INCLUSION • Examples: o Basophilic stippling o Pappenheimer bodies o Howell-Jolly Bodies o Cabot Rings o Heinz Bodies o Hemoglobin H o Malarial stipplings o Babesia spp. o Bartonella spp.

• Basophilic Stippling o Aggregated RNA ▪ Blueberry Bagel Appearance o Punctuate stippling/punctuate basophilia o Seen in: ▪ Lead poisoning (Plumbism) ▪ Arsenic poisoning ▪ Pyrimidine- 5’-nucleotidase deficiency ▪ Anemia with impaired hemoglobin synthesis ▪ Refractory anemias ▪ Alcoholism ▪ Megaloblastic anemia • Pappenheimer Bodies o aka Siderotic granules (Supravital stain) o Intraerythrocytic collections of iron (inside RBC) o Visualization: ▪ Supravital stain: Siderotic granules • Iron stain, Perl’s reagent, Prussian Blue, New methylene blue ▪ Wright stain: Pappenheimer bodies o Seen in: ▪ Sideroblastic anemia ▪ Hemochromatosis ▪ Hemosiderosis • Howell-Jolly Bodies o Remnants of nuclear chromatin (DNA) ▪ In orthochromic normoblast stage, if nucleus is not completely expelled from cell, remnants are produced o Round, solid-staining, dark-blue to purple inclusion (1-2 um) o Visualization: ▪ Wright stain (reddish-blue or purple) ▪ Supravital stain ▪ Feulgen (+) - stains DNA o Seen in: ▪ Megaloblastic anemia ▪ Post-splenectomy • Cabot Rings o Remnants of microtubules from mitotic spindle o Ring-, figure-eight-, loop-shaped inclusion o Visualized using Wright stain (reddish-violet) o Seen in: ▪ Abnormal erythropoiesis ▪ Pernicious anemia ▪ Lead poisoning • Heinz Bodies o Ehrlich bodies o Denatures and precipitated hemoglobin o inclusions, 0.2 to 2.0 um in size o Visualized in crystal violet or brilliant cresyl blue ▪ Cannot be seen in Wright stain o Seen in: ▪ Congenital hemolytic anemia ▪ G6PD Deficiency ▪ Drug-induced hemolytic anemia (phenacetin) ▪ Hemoglobinopathies ▪ Favism - inherited condition associated with sensitivity to fava beans • Hemoglobin H o Abnormal hemoglobin composed of 4 ß-globin chains o Precipitated hemoglobin H. ▪ Pitted-golf ball/raspberry appearance o Visualized using Supravital stain o Seen in Hemoglobin H disease • Malarial Stippling o Fine granular appearance in erythrocytes that harbor malarial Parasites ( Plasmodium spp.)

• Babesia spp. o Inclusion bodies resembling maltese cross • Bartonella spp. o Comma-shaped inclusion bodies • Feulgen - presence of DNA • Supravital - presence of RNA & iron RBC MORPHOLOGY GRADING CHART Morphology Grading • Polychromatophilia • Helmet cells • Dacryocytes • Acanthocytes • Schistocytes • Spherocytes 1+ = 1-5 per field 2+ = 6-10 per field 3+ = >10 per field • Poikolocytosis • Ovalocytes • Elliptocytes • Burr cells • Bizarre-shaped RBC • Target cells • Stomatocytes 1+ = 3-10 per field 2+ = 11-20 per field 3+ = >20 per field • Sickle cells • Basophilic stipplings • Pappenheimer bodies • Howell-Jolly bodies If present = “ POSITIVE ” ABNORMAL ERYTHROCYTE DISTRIBUTION • Rouleaux o Stacking or coining pattern of erythrocytes due to abnormal or increased plasma proteins ▪ “Stack of coins” o Seen in: hyperproteinemia, multiple myeloma, Waldenstrom macroglobulinemia, increased fibrinogen o Artifactual : thicker area of blood smear ▪ The area have clumped RBCs in thicker area o True : seen in thinner area of blood smear ▪ Observed near the feathery edge o Grading : ▪ 1+ - aggregates of 3-4 RBC ▪ 2+ - aggregates of 5-10 RBC ▪ 3+ - many aggregates with only few free RBCs • Agglutination o Clumping of RBC with no pattern o Occurs when RBC are coated with IgM antibodies and complement o Seen in cold autoimmune hemolytic anemia (cold agglutinin disease)