Erythrocyte Abnormalities PDF

MLS 116 - HEMATOLOGY I BSMT-3A ERYTHROCYTE ABNORMALITIES ABNORMAL ERYTHROCYTE MORPHOLOGY ERYTHROCYTE / Red Blood Cells (RBC) Approximate...

MLS 116 - HEMATOLOGY I BSMT-3A ERYTHROCYTE ABNORMALITIES ABNORMAL ERYTHROCYTE MORPHOLOGY ERYTHROCYTE / Red Blood Cells (RBC) Approximately 5 million erythrocytes per microliter of Is found in pathological states that may be abnormalities in: circulating blood. A. Red cell distribution. Primary cell in the blood. B. Size (anisocytosis). C. Hemoglobin content Color Variation. (anisochromia) Lacks a nucleus, has a biconcave shape, and has an average D. Shape (poikilocytosis). volume of 90 fL. E. The presence of inclusion bodies in erythrocytes. Life span - 120 days A. ERYTHROCYTE DISTRIBUTION ABNORMALITIES Use of its biconcave shape: a. Supports deformation b. Enabling the circulating cell to pass smoothly through capillaries where it readily exchanges oxygen and carbon dioxide while contacting the vessel wall. ➔ Cytoplasm - contains abundant hemoglobin (a complex of globin, protoporphyrin, and iron), which transports O2 from the lungs to the tissues. ➔ Hemoglobin has four globin chains, and each chain 1. ROULEAUX FORMATION contains a heme molecule with an iron in the ferrous - Stacking of RBCs due to increased plasma proteins state. This allows each hemoglobin molecule to carry coating RBCs (resembling a stack of coins) four O2 molecules.. RBC also transports: a. Carbon dioxide CO2 b. Bicarbonate (HCO3) - from the tissues back to the lungs. Nutritional requirements Amino acids Vit.B12 Folic Acid Associated with: Hyperfibrinogenemia & Hyperglobulinemia Vitamin B6, Fe2 CHON 2. AGGLUTINATION RECORDING RBC MORPHOLOGY - Antibody-mediated Irregular clumping, it is temperature dependent 1. Scan area using 100x (oil immersion) - Associated with: Cold agglutinin and Warm 2. Observe 10 fields autoimmune hemolysis 3. Red cells are observed for size (anisocytosis), shape (poikilocytosis), hemoglobin content, and the presence or absence of inclusions. 4. Abnormal morphology: Red cell morphology is assessed according to the following sample grading system. Note that red cell morphology must be scanned in a good counting area. (clean and organized) 5. Two questions should be asked - Is morphology seen in every field? - Is the morphology pathologic and not artificially induced? MLS 116 - HEMATOLOGY I BSMT-3A B. VARIATION OF ERYTHROCYTE SIZE - TICS (Thalassemia, Iron-deficiency anemia, chronic (ANISOCYTOSIS) anemia, sideroblastic anemia Variations in size (Microcyte and Macrocyte) a. THALASSEMIA Prominent in severe anemias - Globin chain problem b. IRON-DEFICIENCY ANEMIA 1. NORMOCYTIC RBCs - Less iron c. CHRONIC ANEMIA Normal (Discocyte) size of RBC (8 micrometer) with a range - Inflammation to tissue of 7 to 9 - Damage to tissue 80-100fL - Iron do not enter, decrease RBC Are biconcave and disc shaped and lack a nucleus The d. SIDEROBLASTIC ANEMIA nucleus of a small lymphocyte (8 µm) is a useful guide to the - Immature cells that contain iron deposits size of a red blood cell). - Does not have photophorphyrin IX Others : Lead poisoning 3. MACROCYTIC RBCs insufficient nutrients RBC larger than the normal (>9 micrometer) and is the result of a defect in nuclear maturation or stimulated erythropoiesis. May be round or oval in shape, the diagnostic significance being different. More than 100fL ASSOCIATED WITH: AHA a. APLASTIC ANEMIA - Bone marrow does not produce enough cells. b. HEMOLYTIC ANEMIA - Bone marrow was able to produce normal RBC but factors in blood are destroying RBC. - Ex. Plasmodium falciparum. c. ACUTE BLEEDING - Loss of blood , number of circulating RBC is down. 2. MICROCYTIC RBCs ASSOCIATED WITH: RBC cell smaller than the normal RBC ( ⅓ Pallor area : Paler appearance Inadequate iron source result in a decrease in hemoglobin synthesis Deficient hemoglobin content Decrease in hemoglobin synthesis Lesser hemoglobin < size RBC Cell is Microcytic- Hypochromic red cell Inadequate coloration or a lack of the typical red color ASSOCIATED WITH: associated with an erythrocyte on a peripheral smear. a. IRON DEFICIENCY 3. POLYCHROMASIA Variation in hemoglobin content showing a slight blue tinge (Wright Stain) Gray Blue The polychromatophilic erythrocyte is larger than a mature Erythrocyte If stained with a supravital stain, a polychromatophilic erythrocyte appears to have a thread-like netting within it and is called a reticulocyte. Reticulocytes- appearance may indicate excess production of RBC in the bone marrow, there is anemia increase production in bone marrow. ASSOCIATED WITH: a. THALASSEMIA- Globin chain problem b. IRON DEFICIENCY ANEMIA- Less iron to less anemia; ferrous sulfate is necessary for formation of heme c. CHRONIC ANEMIA-Inflammation to tissue; ASSOCIATED WITH: damage to tissue not enter iron decreases RBC d. SIDEROBLASTIC ANEMIA- Immature cells that a. HEMOLYTIC ANEMIA contain iron deposits Does not have photoporhyrin IX b. BLOOD CANCER POLYCHROMASIA GRADING HYPOCHROMASIA GRADING MLS 116 - HEMATOLOGY I BSMT-3A POIKILOCYTES SECONDARY TO DEVELOPMENTAL MACROCYTOSIS A. OVAL MACROCYTE have an oval or egg-like appearance A.KA megalocyte Although these cells are similar in appearance to elliptocytes, megalocytes are macrocytic and have a fuller and rounder appearance. In contrast, elliptocytes tend to have a normal cell-size volume. - Occurs when MCV reaches 125 fL Bipolar arrangement in hemoglobin Elongated and bigger than normal No central pallor D. RBC VARIATION IN SHAPE (POIKILOCYTOSIS) Mature erythrocytes that have shaped other than the normal ASSOCIATED WITH: round, biconcave appearance on a stained blood smear. Poikilocytes can assume many shapes but frequently a. MEGALOBLASTIC DISORDER resemble common objects such as eggs, pencils, and teardrops.. The names for specific kinds of poikilocytes include: POIKILOCYTE SECONDARY TO MEMBRANE ABNORMALITIES A. ACANTHOCYTES Poikilocytes Secondary to A. Oval macrocyte development A.K.A Spur Cells or Thorn Cells macrocytosis Dark red to salmon, lacking central pallor. Erythrocyte with multiple irregularly spaced spike-like projections often with Poikilocyte secondary to A. Acanthocyte drumstick ends, varying in width, length, and number. membrane abnormalities B. Echinocytes Long spikes C. Codocytes Vary in size 3-12 long spike caused by abnormal ratio of D. Spherocytes Lecithin and Sphingomyelin E. Stomatocyte Acanthocytes are prevalent in two very different disorders: F. Elliptocyte - abetalipoproteinemia, a rare hereditary disorder Poikilocyte secondary to A. Schistocyte - represent an imbalance of erythrocytes and traumatic injury B. keratocyte plasma lipids C. Dacryocyte - The reason for this imbalance is that the patient does not absorb lipids in the small intestine. This results in decreased plasma Poikilocyte secondary to A. Drepanocyte lipids, which in turn produces a membrane abnormal hemoglobin content defect. Other poikilocyte A. Blister cell B. Degmacyte MLS 116 - HEMATOLOGY I BSMT-3A C. CODOCYTES Target Cells/ Mexican Hat Cell Resembles a shooting target mic A central bull’s eye is surrounded by a clear ring then an outer reading. Enzyme defects: - Cholesterol and Phosphatidyl are abnormal increased within erythrocyte and become incorporated - Excessive cholesterol will result to hemoglobin imbalance - Spur cell anemia - Prominent in Thalassemia and Hemoglobinopathies ASSOCIATED WITH: a..ALCOHOL CIRRHOSIS b.HEMOLYTIC ANEMIA c.MALABSORPTION STATES d.HEPATIC HEMANGIOMA e.NEONATAL HEPATITIS f.POSTSPLENECTOMY g.HYPOTHYROIDISM f.VITAMIN E DEFICIENCY B. ECHINOCYTES ASSOCIATED WITH: A.K.A. Crenated Cells and Sea Urchin Cells a. HEMOGLOBINOPATHIES (HB C DISEASE, S-C AND S-S DISEASE Short, scalloped or spike liked projection b. SICKLE CELL THALASSEMIA, AND THALASSEMIA Crenation can occur as the result of the physical loss of c. HEMOLYTIC ANEMIA intracorpuscular water. d. HEPATIC DISEASE WITH JAUNDICE e. SPLENECTOMY Covered with short spikes causes: Hypertonic Solution- when RBC is exposed they will be crenated D. SPHEROCYTES Artifacts- Improperly dried or preparations Deficiency in ATP- changes in osmotic membrane in a cell ; Sphere means ball/round REASON of the presence of SHORT SPIKES No central pallor zone Darker than surrounding red blood cells Loss of membrane and biconcave shape, caused by absence of spectrin. Spherocyte, like erythrocyte may appear as artifacts if a slide is examined at the thin and for the normal blood smear. Maybe formed because of injury or physical trauma ASSOCIATED WITH: a. UREMIA b. PYRUVATE KINASE DEFICIENCY c. MICROANGIOPATHIC HEMOLYTIC ANEMIA d. NEONATES (ESPECIALLY PREMATURE) ASSOCIATED WITH: MLS 116 - HEMATOLOGY I BSMT-3A a..HEREDITARY SPHEROCYTOSIS b. SEVERE BURNS c..BLOOD BAG WAS STORED FOR A LONG TIME ASSOCIATED WITH: d. TRANSFUSED CELLS a..IRON DEFICIENCY Causes of spherocyte formation: b. THALASSEMIA 1.Microspherocyte c. HEMOLYTIC ANEMIA 2. Hereditary Spherocytosis d. PERNICIOUS ANEMIA e. SICKLE CELLS TRAIT E. STOMATOCYTES f. HEMOGLOBIN (HB) C DISEASE g. HEREDITARY STOMATOCYTOSIS Mouth cell or Bowel shaped cell h..LIVER DISEASE - Have a slit opening that resembles mouth i. RH NULL PHENOTYPE Caused by osmotic exchange due to cation imbalance in red cells. Stomatocytes result from increased sodium (Na+) ion and decreased potassium (K+) ion concentrations within the POIKILOCYTES SECONDARY TO TRAUMA AND INJURY cytoplasm of the erythrocyte. Increased permeability to sodium causing improper or imbalance distribution of A.SCHISTOCYTES hemoglobin in RBC Are fragments of erythrocytes that are small and irregularly shaped: Deeper red appearance Produce Fragmentation Forms due to blood vessel injury Fragmentation by damage of RBC Increased numbers of schistocytes can be seen in hemolytic anemias related to burns and prosthetic implants as well as renal transplant rejections. Fragmentation caused by: 1. Clot ASSOCIATED WITH : 2. Prosthetic Heart Valve 3. Altered Heart Vessels a. ALCOHOLISM B. CIRRHOSIS C. GLUTATHIONE DEFICIENCY D. HEREDITARY SPHEROCYTOSIS E. INFECTIOUS MONONUCLEOSIS F. LEAD POISONING MALIGNANCIES G. THALASSEMIA MINOR H. TRANSIENTLY ACCOMPANYING HEMOLYTIC ANEMIA. -These cells can also be seen in hereditary stomatocytosis and Rh null disease, which both lack the Rh antigen complex. ASSOCIATED WITH: F. ELLIPTOCYTES a. MICROANGIOPATHIC HEMOLYTIC ANEMIA Elongated and narrow; Rod and Cigar, Sausage shaped b.HEMOLYTIC UREMIC SYNDROME They represent a membrane defect in which the membrane is c.THROMBOTIC THROMBOCYTOPENIC PURPURA radically affected and suffers a loss of integrity. d.DISSEMINATED INTRAVASCULAR Defects on cytoskeleton COAGULATION Defect in protein band 4-1 resulting in elongation e.SEVERE BURNS, RENAL GRAFT REJECTION B. KERATOCYTES MLS 116 - HEMATOLOGY I BSMT-3A Erythrocytes that are partially deformed but One of the end of the cell is pointed not cut. - ( Results from gelation of polymerized deoxygenated Schistocytes with horn like projections can be encountered Hbs The spicules, resembling two horns, result from a ruptured - Lower oxygen levels lower the pH ) vacuole. Usually, the cell appears like a half-moon or The influx of sodium ions and other metabolic changes spindle. These cells are seen in conditions such as produce an extremely increased level of intracellular calcium disseminated (diffuse) intravascular coagulation (DIC). ions. Alterations in the cellular contents produce cell membrane rigidity. The presence of sickle is associated with sickle cell anemia. - Sickling is due to the precipitation of hemoglobin ASSOCIATED WITH: A. SICKLE CELL ANEMIA B. HEMOGLOBIN S CELLS- ( ABNORMAL AMINO ACID IN GLOBIN) ASSOCIATED WITH: a. MICROANGIOPATHIC HEMOLYTIC ANEMIA b. BURNS c.DISSEMINATED INTRAVASCULAR COAGULOPATHY C. DACRYOCYTES OTHER POIKILOCYTES Resembles tears/pear ( Tear Drop Cell);may have one blunt A. BLISTER CELLS projection Are erythrocytes containing one or more Usually smaller than normal erythrocytes due to squeezing vacuoles that resemble a blister on the skin. and fragmentation Precursors cells before turning schizocyte Usually a larger scooped out part of the cell Blister cells result from the traumatic interaction of blood vessels and circulating blood such as fibrin deposits. The vacuoles may rupture( schizocyte and keratocytes can be seen) ASSOCIATED WITH: a. SPLENECTOMY b. THALASSEMIA c. MYELOID DYSPLASIA ASSOCIATED WITH: d. HOMOZYGOUS BETA THALASSEMIA e. PERNICIOUS ANEMIA a. INCREASED IN PULMONARY EMBOLI IN SICKLE f. SEVERE ANEMIA CELL ANEMIA b. MICROANGIOPATHIC HEMOLYTIC ANEMIA POIKILOCYTE SECONDARY TO ABNORMAL Hb CONTENT A. DREPANOCYTE ( SICKLE CELLS) B. DEGMACYTE ( BITE CELLS) Resemble a crescent/ leaf like MLS 116 - HEMATOLOGY I BSMT-3A Denature hemoglobin, the inclusion bodies, Heinz bodies will be removed by the macrophages in the spleen Bile marks are permanently damage in your red cell PARASITES OF RBC All 4 species in malaria and Babesia microti GRADING OF RBC INCLUSIONS ASSOCIATED WITH: a.G6PD- AFTER OXIDANT-RELATED HEMOLYSIS. b. THALASSEMIA c. DRUG INDUCED ANEMIA GRADING OF RBC POIKILOCYTES (OIL IMMERSION) REPORTING OF POIKILOCYTE Slight 15% REPORTING OF RESULTS PARTICULAR VARIATION Occasional 10% normochromic, report out as NORMAL. When abnormal morphology has been noted, DO NOT indicate normal on the report form. EXAMPLE: 7-10 microcytic RBC's/OIF is reported out as: 2+ microcytosis or Moderate microcytos STAINING CHARACTERISTIC MLS 116 - HEMATOLOGY I BSMT-3A LESSON 8: ERYTHROCYTE PRODUCTION AND DESTRUCTION ERYTHROID PROGENITOR NORMOBLAST refers to developing nucleated RBC precursors (i.e., blasts) with normal appearance. PRONORMOBLAST morphologically identifiable erythrocyte precursors develop from two progenitors, BFU-E) and (CFU-E), both committed to the erythroid cell line. Burst-forming Unit-Erythroid SUMMARY earliest committed progenitor came from pluripotent stem cells gives rise to large colonies because they are capable of multisubunit colonies (called bursts), it takes about 1 week for the BFU-E to mature to the CFU-E and another week for the CFU-E to become a pronormoblast, Colony Forming Unit give rise to smaller colonies. cell completes approximately three to five divisions before maturing further Overall: - It takes 6-7 days- maturation of precursor to enter the circulation approx. 18-21 days are required to produce a mature RBC from the BFU-E. →pluripotent stem cells→ BFU-E →CFU-E →Pronormoblast ERYTHROID PRECURSOR Normoblastic proliferation is a process encompassing replication to increase cell numbers and development from immature to mature cell stages In the erythrocyte cell line, there are typically three and occasionally as many as five divisions 8 to 32 mature RBCs usually result MLS 116 - HEMATOLOGY I BSMT-3A PRONORMOBLAST (RUBRIBLAST) 8:1 *Round to oval with 1 or 2 nucleoli *Purple red chromatin is open and contains few Cytoplasm is dark blue because of the concentration of ribosomes and RNA Golgi complex may be visible next to the nucleus as a pale, unstained area Show small tufts of irregular cytoplasm along the periphery of the membrane Undergoes mitosis and gives rise to two daughter pronormoblasts. More than one division is possible before maturation into basophilic normoblasts. Present only in the bone marrow Begins to accumulate the components necessary for hemoglobin production. Proteins and enzymes necessary for iron uptake and protoporphyrin synthesis are produced Globin production begin Length of time on this stage; 24 hours The most important features in the identification of RBCs are: 1. the nuclear chromatin pattern (texture, density, homogeneity) 2. nuclear diameter, nucleus-to-cytoplasm (N:C) ratio presence or absence of nucleoli 3. cytoplasmic color Nucleus-to-cytoplasm (N:C) ratio BASOPHILIC NORMOBLAST (PRORUBRICYTE) - is a morphologic feature used to identify and 6:1 stage red blood cell and white blood cell chromatin begins to condense precursors. parachromatin areas become larger and sharper, and - estimate of the area of the cell occupied by the N:C ratio decreases the nucleus compared with that of the chromatin stains deep purple-red cytoplasm Nucleoli may be present early in the stage but disappear late cytoplasm may be a deeper, richer blue than in the pronormoblast undergoes mitosis, giving rise to two daughter cells. More than one division is possible before the daughter cells mature into polychromatic normoblasts present only in the bone marrow Detectable hemoglobin synthesis occurs many cytoplasmic organelles, including ribosomes and substantial amount of messenger ribonucleic acid completely mask the minute amount of hemoglobin pigmentation - LOT: 24hrs MLS 116 - HEMATOLOGY I BSMT-3A present only in the bone marrow Hemoglobin production continues on the remaining ribosomes using messenger RNA Late in this stage, the nucleus is ejected from the cell loss of vimentin ( a protein responsible for holding organelles in proper location in the cytoplasm) is probably important in the movement of the nucleus to the cell periphery *nucleus-containing projection separates from the cell by having the membrane seal and pinch off the projection Nonmuscle myosin of the membrane is important in this POLYCHROMATIC NORMOBLAST (RUBRICYTE) pinching process. macrophages recognize phosphatidylserine on the 4:1 (1:1 at end of this stage) pyrenocyte surface as an “eat me” flag no nucleoli are present. small fragments of nucleus are left behind if the projection first stage in which the pink color associated with is pinched off before the the entire nucleus is enveloped. stained hemoglobin can be seen. These fragments are called Howell-Jolly bodies. They are stained color reflects the accumulation of hemoglobin pigmentation over time and concurrent decreasing typically removed from the cells by the splenic amounts of macrophage pitting process once the cell enters the RNA is a mixture of pink and blue, resulting in a circulation. murky gray-blue. LOT: 48hrs polychromatophilic means “many color loving. last stage in which the cell is capable of undergoing mitosis present only in the bone marrow Hemoglobin synthesis increases accumulation begins to be visible as a pinkish color in the cytoplasm. RNA and organelles are still present, particularly ribosomes, which contribute a blue color to the cytoplasm. progressive condensation of the nucleus and disappearance of nucleoli are evidence of progressive decline in transcription of deoxyribonucleic acid (DNA) POLYCHROMATIC ERYTHROCYTE (RETICULOCYTE) LOT: 30hrs N0 nucleus ORTHOCHROMIC NORMOBLAST ( METARUBRICYTE) predominant color is that of hemoglobin yet with a bluish tinge due to some residual ribosomes and RNA Lacking a nucleus, the polychromatic erythrocyte cannot divide. resides in the bone marrow for about 1 to 2 days and then moves into the peripheral blood for about 1 day before reaching maturity first several days after exiting the marrow, the polychromatic erythrocyte is retained in the spleen for pitting of inclusions and membrane polishing by splenic macrophages, which results in the biconcave discoid mature RBC 1:2 The nucleus is completely condensed (i.e.,pyknotic completes production of hemoglobin from a small amount The increase in the salmon pink color of the cytoplasm of residual messenger RNA using the remaining ribosomes. reflects nearly complete hemoglobin production. -Endoribonuclease digests the ribo somes. A small amount RNA reacts with the basic component of the stain and of residual ribosomal RNA is present, however can be contributes a slightly bluish hue to the cell but that fades visualized with a vital stain such as new methylene blue toward the end of the stage as the RNA and organelles are called reticulocyte when stained with vital stain degraded. (methylene blue) not capable of division because of the condensation of the chromatin MLS 116 - HEMATOLOGY I BSMT-3A The reticulocyte is called a polychromatic erythrocyte central pallor is about one-third the diameter of the because it lacks a nucleus and is no longer an erythroblast cell but still has a bluish tinge. erythrocyte cannot divide the name reticulocyte is often used to refer to the stage Mature RBCs remain active in the circulation for 120 immediately preceding the mature erythrocyte days LOT: 24 -48 hrs or 3 days; mature erythrocyte delivers oxygen to tissues, releases it, and returns to the lung to be reoxygenated 2 days (spent in the bone marrow) third (spent in the It contains mostly hemoglobin, the oxygen-carrying peripheral blood or sequestered in the spleen component. its membranes are flexible and deformable, that is, able to flex but return to its original shape. RBCs must squeeze through small spaces such as the basement membrane of the bone marrow venous sinus ERYTHROKINETICS term describing the dynamics of RBC production and destruction Erythron- name given to the collection of all stages of erythrocytes throughout the body: conveys the concept of a unified functional tissue erythron is the entirety of erythroid cells in the distinguished from the RBC mass. RBC mass refers only to the cells in circulation. Hypoxia the stimulus to RBC production primary oxygen-sensing system of the body is located in peritubular fibroblasts of the kidney too little tissue oxygen, is detected by the peritubular fibroblasts, which then produce erythropoietin (EPO), the major stimulatory cytokine for RBCs Hemorrhage; EPO is increased Hypoxia induced rbc production is regulated by a family of transcription factor proteins, called hypoxia inducible factors (HIFs) HIFs respond to hypoxia by binding to kidney hypoxia ERYTHROCYTE responsive elements located at the 5’ flanking region of the EPO gene. This results in increased EPO gene No nucleus transcription, EPO production, and ultimately increased a biconcave disc; 7 to 8 mm in diameter; thickness of RBC Production about 1.5 to 2.5 mm. kidney is the body’s hypoxia sensor and provides early appears salmon-pink stained cell with a central pale detection when oxygen levels decline area that corresponds to the concavity MLS 116 - HEMATOLOGY I BSMT-3A an antiapoptotic molecule produces when activated JAK2 phosphorylates the STAT 5 pathway ERYTHROPOIETIN EPO-stimulated cells develop this molecule on their mitochondrial membranes, preventing release of primary hormone that stimulates the production of cytochrome c erythrocyte EPO is a thermostable, nondialyzable, glycoprotein Cytochrome C- apoptotic initiator hormone with a molecular weight of 34 kD consists of a carbohydrate unit and a terminal sialic *EPO’s effect is mediated by the transcription factor, GATA1, which acid unit is essential to red cell survival a true hormone, being produced at one location (kidney) and acting at a distant location (bone Reduced Marrow Transit Time marrow). growth factor (or cytokine) that initiates an intracellular EPO stimulates the synthesis of RNA in erythroid message to the developing erythroid cells; this precursors and effectively increases the rate of the process is called signal transduction. developmental process. - steady state↓ Hypoxia ↑ Among the processes that are accelerated is hemoglobin interaction of EPO with its receptor initiates a cascade of production. intracellular events that ultimately leads to increased cell EPO induces erythroid precursors to secrete division and maturation, increased intestinal iron erythroferrone absorption and hemoglobin synthesis, and more RBCs entering the circulation. Erythroferrone Janus Activated tyrosine kinase 2 (JAK2) - acts on hepatocytes to decrease hepcidin production - allows more iron to be absorbed from the intestines to signal transducers that are associated with the cytoplasmic support the increased hemoglobin synthesis domains of the EPO receptor. another accelerated process is bone marrow egress as a activates downstream signal transduction pathways that result of the loss of adhesive receptors (such as the ultimately promotes transcription of specific genes in the fibronectin receptor discussed later) and the acquisition of RBC nucleus egress-promoting surface molecules. EPO has three major effects: Also accelerated the cessation of cell division 1. allowing early release of reticulocytes from the bone marrow 2. preventing apoptotic cell death MEASUREMENT OF ERYTHROPOIETIN 3. reducing the time needed for cells to mature in the bone marrow. EPO measured by chemiluminescence Quantitative measurements of EPO are performed on Preventing apoptotic cell death plasma and other body fluids. reference interval: 10 to 30 U/L sufficient to maintain Apoptosis is the mechanism by which an appropriate steady-state erythropoiesis in a healthy adult. normal production level of RBCs is controlled. expected ↑ EPO in urine on anemia px except px with Fas, the death receptor, is expressed by young erythroid renal disease anemia precursors FasL, the ligand, is expressed by older erythroid THERAPEUTIC USES OF ERYTHROPOIETIN precursors. As long as older cells mature slowly in the marrow, they induce the death of unneeded younger cells. athletes illicitly use EPO injections to increase the CFU-E has the most EPO receptors and is most sensitive to oxygen-carrying capacity of their blood to enhance EPO rescue endurance and stamina, especially in long-distance When EPO binds to its receptor on the CFU-E, one of the running and cycling. effects is to reduce production of Fas ligand. one of the methods of blood doping; Without EPO, the CFUE does not survive. Bcl-XL (now called Bcl-2-like protein 1) MLS 116 - HEMATOLOGY I BSMT-3A This pathway accounts for a minor component of OTHER STIMULI TO ERYTHROPOIESIS normal destruction of RBCs. Testosterone directly stimulates erythropoiesis, which partially explains the higher hemoglobin concentration in men than in women. Pituitary and thyroid hormones have been found to affect the production of EPO and so have indirect effects on erythropoiesis. MICROENVIRONMENT OF THE BONE Erythropoiesis typically occurs in what are called erythroid islands within the bone marrow These islands consist of a central macrophage surrounded by erythroid precursors in various stages of development As erythroid precursors mature, they lose adhesive molecule receptors, which allows their egress from the bone marrow. Egress occurs between adventitial cells but through pores in the endothelial cells of the venous sinus Erythroid precursors would not survive without macrophage support via such stimulation. The major cellular anchor for the developing normoblasts is the macrophage ERYTHROCYTE DESTRUCTION All cells experience deterioration of their enzymes over time because of natural catabolism. Because RBCs lack mitochondria, they rely on glycolysis for production of adenosine triphosphate (ATP). loss of glycolytic enzymes is central to this process of cellular aging, called senescence Senescence culminates in phagocytosis by macrophages. This is the major method by which RBCs die normally. MARROW MACROPHAGE-MEDIATED HEMOLYSIS accounts for most normal RBC deaths. The signals to macrophages that initiate RBC ingestion may include binding of autologous immunoglobulin G (IgG), expression of phosphatidylserine on the outer membrane, cation balance changes, and binding of CD47 to thrombospondin-1. MECHANICAL HEMOLYSIS (FRAGMENTATION OR INTRAVASCULAR HEMOLYSIS) intravascular rupture of RBCs from purely mechanical or traumatic stress results in fragmentation and release of the cell contents into the blood (peripheral circulation) MLS 116 - HEMATOLOGY I BSMT-3A MLS 116 - HEMATOLOGY I BSMT-3A LESSON 9: ERYTHROCYTE METABOLISM After its lifeline RBC is disassembled into its reusable components: AND MEMBRANE STRUCTURE FUNCTION a. globin chains and iron from hemoglobin b. phospholipids and proteins from the cell membrane TOPIC OUTLINE A. Energy Production—Anaerobic Glycolysis The protoporphyrin ring of hemoglobin is not reusable and is excreted as bilirubin. B. Glycolysis Diversion Pathways (Shunts) Hexose Monophosphate Pathway Methemoglobin Reductase Pathway A. ENERGY PRODUCTION—ANAEROBIC GLYCOLYSIS Rapoport-Luebering Pathway Without the Mitochondria, RBC relies on anaerobic glycolysis C. RBC Membrane RBC Deformability for its energy. Exchange of O2 and CO2 is a passive function RBC Membrane Lipids from high partial pressure to low partial pressure. RBC Membrane Proteins Osmotic Balance and Permeability Cells’ metabolic processes requiring energy: DEFINITION OF TERMS 1. Cyanosis - is a blue skin coloration that occurs when the blood does not deliver enough oxygen to the tissues. It is a common sign of heart or lung disease, in which the blood fails to become oxygenated or is distributed improperly throughout the body. 2. Methemoglobin - hemoglobin in the form of metalloprotein, in which the iron in the heme group is in the Fe³⁺ state, not the As energy production slows, the RBC grows senescent and is Fe²⁺ of normal hemoglobin. Sometimes, it is also referred to as removed from the circulation. ferrihemoglobin. Methemoglobin cannot bind oxygen, which means it cannot carry oxygen to tissues. Hereditary nonspherocytic hemolytic anemia - a hereditary deficiencies of nearly every glycolytic enzyme. Their common Methemoglobin reductase is also called cytochrome b5 result is shortened RBC survival. reductase. Embden-Meyerhof Pathway (EMP) or Anaerobic Pathway 3. Idiopathic Methemoglobinemia - a very rare blood disorder, of Glucose Metabolism requires glucose to generate ATP, a sometimes called “blue baby syndrome,” which affects how red high-energy phosphate source. blood cells deliver oxygen to cells and tissues. RBCs lack internal energy stores and rely on plasma glucose to enter the cell to generate ATP. ERYTHROCYTE PRODUCTION Glucose enters the RBC through facilitated diffusion via the Produced through normoblastic proliferation and mature in transmembrane protein. Glucose is then catabolized to the bone marrow. pyruvate (pyruvic acid) in the EMP. The nucleus, present in maturing normoblasts, is extruded as part of the RBC maturation process. Generated ATP : 4 molecules Cytoplasmic ribosomes and mitochondria also disappear 24 Net gain - 2 molecules to 48 hours Three phases of Glycolysis FIRST PHASE: Employs glucose phosphorylation, NOTE: isomerization, and diphosphorylation to yield fructose 1, Without mitochondria for aerobic respiration via oxidative 6-bisphosphate (F1,6-BP). Fructose-bisphosphate aldolase phosphorylation, adenosine triphosphate (ATP) is produced then cleaves F1,6-BP to produce glyceraldehyde3-phosphate. within the cytoplasm through anaerobic glycolysis (Embden-Meyerhof pathway, EMP) for the lifetime of the cell. MLS 116 - HEMATOLOGY I BSMT-3A In this phase, 2 molecules of ATP were utilized. DEFINITION OF TERMS - ( memory refresher only!) B. GLYCOLYSIS DIVERSION PATHWAYS (SHUNTS) Phosphorylation is the process of adding a phosphate group (PO₄³⁻) to a molecule, typically from ATP. This is a Three alternate pathways, called diversions or shunts, common way to activate or deactivate enzymes and other branch from the glycolytic pathway. The three diversions are molecules, making them more reactive. In glycolysis, glucose undergoes phosphorylation to form the hexose monophosphate pathway (HMP), the glucose-6-phosphate. methemoglobin reductase pathway, and the Rapoport-Luebering pathway. Isomerization is the rearrangement of the molecular structure of a compound without adding or removing any atoms, converting it into another isomer. For example, in SIDE NOTE (for better understanding) glycolysis, glucose-6-phosphate is isomerized into fructose-6-phosphate. Basically, these diversion paths kay mura ra siya’g mga continuation sa mga naagian ni EMP. So in short, ang whole Diphosphorylation refers to the addition of two phosphate pathway ani kay connected ra sila tanan. groups to a molecule. In glycolysis, fructose-6-phosphate is diphosphorylated to form fructose 1,6-bisphosphate GDP are alternative routes within the glycolytic pathway that (F1,6-BP), which has two phosphate groups attached at allow cells to produce additional molecules necessary for different positions. specific functions, without following the standard glycolytic process from glucose to pyruvate. I’ll put the diagram (from the book) after nako’g introduce sa SECOND PHASE: Converts Glyceraldehyde 3-phosphate 3 ka diversion pathways, so pwede ninyo i check sa next (G3P) to 3-phosphoglycerate (3-PG). page. LABAN FUTURE RMTs!! This phase generates two ATP molecules and 3-PG. These 3 pathways are critical for RBC function, ensuring that the cells can manage oxidative stress, maintain effective oxygen transport, and regulate oxygen release depending on the body's needs. 1. Hexose Monophosphate Pathway Process: THIRD PHASE: The third phase of glycolysis converts 3-PG to a. Glucose-6-Phosphate (G6P) Diversion: pyruvate. Glucose-6-phosphate (G6P) is diverted from glycolysis into the HMP pathway. Pyruvate may diffuse from the erythrocyte or may become a substrate for lactate dehydrogenase (LD or LDH) with b.Conversion to 6-Phosphogluconate (6-PG): regeneration of the oxidized form of nicotinamide adenine G6P is oxidized to 6-phosphogluconate (6-PG) by the dinucleotide (NAD1). enzyme glucose-6-phosphate dehydrogenase (G6PD). This phase generates 2 ATP molecules. During this step, NADP⁺ is reduced to NADPH. c. NADPH Production: NADPH produced in the previous step is used to reduce oxidized glutathione (GSSG) to its active form, reduced glutathione (GSH), via the enzyme glutathione reductase. MLS 116 - HEMATOLOGY I BSMT-3A d. Detoxification of Peroxides: Thus, the MRP relies on the NADH generated during the EMP's glycolytic process to function effectively. Reduced glutathione (GSH) then detoxifies hydrogen peroxide (H₂O₂) by converting it to water and oxygen, using the Process: enzyme glutathione peroxidase. a.Oxidation: This process protects RBCs from oxidative damage. Heme iron in hemoglobin is oxidized from Fe²⁺ to Fe³⁺ by e. Further Catabolism of 6-PG: exposure to oxygen and peroxides, forming methemoglobin, which cannot carry oxygen. 6-PG is further catabolized to ribulose 5-phosphate (R5P), carbon dioxide (CO₂), and another molecule of NADPH by the b. Reduction: enzyme 6-phosphogluconate dehydrogenase. Methemoglobin is reduced back to its functional Fe²⁺ state by cytochrome b5 reductase, using electrons from NADH f. Final Products: (produced in glycolysis) as an intermediate carrier. The pathway produces NADPH, ribulose 5-phosphate (R5P), and CO₂, which are crucial for RBCs to maintain their function Importance: and structure under oxidative stress. Restores hemoglobin’s ability to carry oxygen, ensuring Importance: efficient oxygen transport in RBCs. Protects RBCs by detoxifying harmful peroxides. Cytochrome b5 reductase handles the majority (over 65%) of methemoglobin reduction, maintaining hemoglobin in its Maintains hemoglobin and cell membrane integrity. functional form. Critical for RBCs to handle oxidative stress. Key Details: Key Details: Methemoglobin: Non-functional form of hemoglobin with Fe³⁺. HMP: Diverts G6P, generates NADPH. Cytochrome b5 Reductase: Key enzyme in reducing NADPH: Reduces GSSG to GSH. methemoglobin back to Fe²⁺. GSH: Detoxifies H₂O₂, protects RBCs. NADH: Provides the necessary electrons for the reduction process. G6PD Deficiency: Leads to vulnerability to oxidative damage and hereditary nonspherocytic anemia. 3. Rapoport-Luebering Pathway Process: a. Diversion: 1,3-Bisphosphoglycerate (1,3-BPG) is diverted from glycolysis to form 2,3-Bisphosphoglycerate (2,3-BPG) by the enzyme bisphosphoglycerate mutase. b.Function of 2,3-BPG: 2,3-BPG binds to hemoglobin, stabilizing it in the 2. Methemoglobin Reductase Pathway deoxygenated state (tense state). This binding shifts the hemoglobin-oxygen dissociation curve to the right, enhancing The Methemoglobin Reductase Pathway (MRP) interacts with oxygen delivery to tissues. the Embden-Meyerhof Pathway (EMP) specifically at the stage where NADH is generated. This occurs during the conversion c. Conversion Back: of glyceraldehyde-3-phosphate (G3P) to 2,3-BPG is then converted to 3-Phosphoglycerate (3-PG) by 1,3-bisphosphoglycerate (1,3-BPG) in the EMP. bisphosphoglycerate phosphatase. This conversion sacrifices the production of two ATP molecules. Detailed Connection: Importance: Step in EMP: The conversion of G3P to 1,3-BPG is catalyzed by the enzyme glyceraldehyde-3-phosphate dehydrogenase Oxygen Delivery: By stabilizing hemoglobin in the (G3PD), which reduces NAD⁺ to NADH. deoxygenated state, 2,3-BPG facilitates the release of oxygen to tissues, which is essential under low oxygen conditions. Entry Point for MRP: The NADH produced in this step is then utilized by the Methemoglobin Reductase Pathway to reduce ATP Balance: The diversion leads to an ATP deficit due to the methemoglobin (Fe³⁺) back to hemoglobin (Fe²⁺). loss of ATP production at both the 1,3-BPG and pyruvate kinase (PK) steps. The cell must balance energy production MLS 116 - HEMATOLOGY I BSMT-3A with the need to regulate hemoglobin oxygen affinity. Key Details: 2,3-BPG: Stabilizes hemoglobin in the deoxygenated state, enhancing oxygen release. Bisphosphoglycerate Mutase: Converts 1,3-BPG to 2,3-BPG. ATP Deficit: Diverting 1,3-BPG to 2,3-BPG results in the loss of ATP production. gj Regulation: Acidic pH and low levels of 3-PG and 2-PG inhibit bisphosphoglycerate mutase, reducing 2,3-BPG levels and favoring ATP generation by reverting 2,3-BPG to 3-PG. C. RBC MEMBRANE RED BLOOD CELL DEFORMABILITY Biconvave Average 90fL 140um surface area Shape enables them to stretch as they pass through narrow capillaries and splenic spores at 2 um in diameter (when MCHC is greater than its normal range and it shortens Semipermeable lipid bilayer supported by the mesh its RBC life span) like protein cytoskeleton 5um thick plasmalemma;100 times more Proteins and Phospholipids are organized elastic asymmetrically - RBC are complex, metabolically active cells using Biochemical composition includes 52% proteins, glucose to make ATP and reducing equivalents to 40% lipids and 8% carbohydrates.. ensure flexibility and O2 delivery - RBC Deformability depends on Relative Cytoplasmic Viscosity - Mean Cell Hemoglobin Concentration(MCHC) Normal RBC MEMBRANE LIPIDS Range - 32%- 36% - MCHS rises Internal Viscosity rises( directly Choline containing, uncharged phospholipids, outer proportional) layer: - 120 days - LIFE SPAN ○ Phosphatidylcholine(PC) (30%) - Neither use O2 for extraction of energy nor ○ Sphingomyelin(SM) (25%) synthesizes protein - Proteomics has identified 2200 separate proteins in RBC that are product of 5% of all human genes Charged phospholipids, inner layer: C. RED BLOOD CELL MEMBRANE ○ Phosphatidylethanolamine (PE) (28%) ○ Phosphatidylserine(PS)(14%) - apoptotic Erythrocyte membrane that is normal in structure and function marker of RBC is essential for survival of red cells. Accounts for the cell’s antigenic characteristics Maintains stability and normal discoid shape of cell Preserve cell deformability Retain selective permeability MLS 116 - HEMATOLOGY I BSMT-3A Asymmetric phospholipids distribution in maintained by: ○ Differential rate of diffusion through membrane bilayer of choline containing phospholipids( PC and SM diffuse slowly) ○ Charged phospholipids interaction with membrane skeletal protein. ○ Active transport of amino phospholipids(PC and PE) from outer to inner layer. This asymmetric phospholipid distribution among the bilayer is the result of the function of several energy-dependent and energy independent phospholipid transport proteins. RBC MEMBRANE PROTEINS Integral Proteins - Embedded in membranes via hydrophobic interactions with lipids. Peripheral protein ○ Located on the cytoplasmic surface of lipid bilayer, it constitutes a membrane skeleton. ○ Anchored via integral proteins. ○ Responsible for membrane elasticity and stability. MLS 116 - HEMATOLOGY I BSMT-3A INTEGRAL PROTEINS Human RBCs glycophorins are integral membrane proteins rich in sialic acids that carry blood group antigenic Band 3 determinants and serve as ligands for viruses and parasites. Glycophorin Aquaporin AQUAPORINS Aquaporins selectively conduct water molecules in and out of the cell, while preventing the passage of BAND 3 ions and other solutes. Allow RBC to remain in osmotic equilibrium with Functions: extracellular fluid ○ Anion transport Exchange bicarbonate for chloride Structural: ○ LInkage of lipid bilayer to underlying membrane skeleton Interaction with ankyrin and protein 4.2, secondarily through binding to protein 4.1 Important for prevention of surface loss. GLYCOPHORINS Comprising 2% of RBC membrane proteins. ○ Sialic acid rich glycoproteins( A,B,C) 3 Domains: - Cytoplasmic - TRansmembrane: Single Spanning alpha helix - Extracellular: glycosylated MLS 116 - HEMATOLOGY I BSMT-3A ○ Alpha and Beta, entwined to form dimers. ○ Associate head to head to form tetrameters. End to end association of these tetrameters with short actin filaments produces the hexagonal complexes observed. ACTIN Short, uniform filaments 35 nm in length Length modulated by tropomyosin/ tropomodulin Spectrintail associated with actin filaments Approx 6 spectrin ends interface with one actin filament, stabilized by protein 4.1 ANKYRIN Interacts with band 3 and spectrin to achieve linkage between bilayer and skeleton Augmented by protein 4.2 PROTEIN 4.1 Stabilizes actin-spectrin interactions ADDUCIN Also stabilizes interaction of spectrin with actin Influenced by calmodulin (thus can promote PERIPHERAL MEMBRANE PROTEINS spectrin-actin interactions as regulated by intracellular Ca concentration) Spectrin Actin TROPOMODULIN Protein 4.1 Paladin ( band 4.2) Caps actin filament Ankyrin Adducin TROPOMYOSIN Tropomyosin Tropomodulin Stabilizes and regulates actin polymerization SPECTRIN Flexible, rod-like molecules, 100 nm length. Responsible for biconcave shape of RBC Two subunits: MLS 116 - HEMATOLOGY I BSMT-3A PERMEABILITY: Permeability properties of the RBC membrane and the active RBC cation transport prevent colloid hemolysis and control the volume of RBC. Freely permeable to water and anions; relatively impermeable to cations. CHARACTERISTICS OF RBCs When RBC are ATP depleted Ca and Na are allowed to accumulate intracellularly and K and water are lost. DEFORMABILITY: METABOLIC PATHWAYS It is controlled by an ATP driven membrane cytoskeleton. Mainly anaerobic, RBC have to deliver not consume Loss of ATP leads to decrease in phosphorylation of oxygen.. spectrin. No nucleus/No mitochondria. Increase in deposition of membrane calcium. RBC metabolism may be divided into anaerobic Rigid cells are removed from the circulation glycolysis and 3 ancillary pathways RBCs contain no mitochondria, so there is no respiratory chain, no citric acid cycle, and no oxidation of fatty acids or ketone bodies. The RBC is highly dependent upon glucose as its energy source. Energy in the form of ATP is obtained ONLY from the glycolytic breakdown of glucose with the production of lactate (anaerobic glycolysis). RBC METABOLISM Glucose transport through RBC membrane: MLS 116 - HEMATOLOGY I BSMT-3A ○ Glucose is transported through the RBC GLYCOLYSIS DIVERSION PATHWAYS (SHUNTS) membrane by facilitated diffusion through glucose transporters (GLUT-1). - three alternative pathways that branch from the glycolytic pathway GLYCOLYSIS 1. Methemoglobin Reductase Pathway Importance of glycolysis in red cells: 2. Hexose Monophosphate Pathway hway or Aerobic Energy production: It is the only pathway that Glycolysis supplies the red cells with ATP. Reduction of methemoglobin: Glycolysis provides 3. Rapoport-Luebering Pathway NADH for reduction of metHb by NADH- cytb5 reductase UTILIZATION OF ATP In red cells 2,3 bisphosphoglycerate binds to Hb, decreasing its affinity for O2, and helps its availability Phosphorylation of sugars and proteins to tissues. ATPase driven ion pumps Maintenance of membrane asymmetry Maintenance of red cell shape and deformability using ATP dependent cytoskeleton METH Hb REDUCTASE PATHWAY Maintains Iron in reduced state for effective transport of O2 Protect SH group of Hb and membrane proteins from oxidation MLS 116 - HEMATOLOGY I BSMT-3A LEUBERING RAPOPORT SHUNT The hydrogen ion concentration is the most important physiological modulator Binding of 2,3 DPG to DeoxyHb stabilize the tense state of Hb and favours release of O2 Free 2,3 DPG also binds with Band 3 and causes partial detachment of membrane from cytoskeleton HEMOGLOBIN OXYGEN DISSOCIATION allowing lateral movement of membrane structure Dissociation and binding of oxygen by hemoglobin are PENTOSE PHOSPHATE PATHWAY not directly proportional to pO2. aka HEXOSE MONOPHOSPHATE PATHWAY Sigmoid-curve. Production of NADPH - 'reducing power' It permits a considerable amount of O2 to be Glutathione is needed in reduced form for: delivered to the tissues with a small drop in O2 ○ Elimination of peroxide tension. ○ Protection of proteins SH groups This shunt also provide ribose 5 phosphate needed for PRPP (substrate for adenine nucleotides reqd for continuing ATP synthesis) MLS 116 - HEMATOLOGY I BSMT-3A SUMMARIZED CONTENT FOR ERYTHROCYTE METABOLISM AND MEMBRANE STRUCTURE AND FUNCTION Glucose enters the red blood cell (RBC) by facilitated diffusion via the transmembrane protein Glut-1. The anaerobic Embden-Meyerhof pathway (EMP) OSMOTIC BALANCE AND PERMEABILITY metabolizes glucose to pyruvate, consuming two adenosine triphosphate (ATP) molecules. The EMP RBC Membrane IMPERMEABLE : Na+, K+ and Ca2+ subsequently generates four ATP molecules per RBC Membrane PERMEABLE : Water, HCO3- and glucose molecule, a net gain of two. Cl- The hexose-monophosphate pathway (HMP) AQUAPORIN 1 - cause for internal osmotic changes converts glu- cose to pentose and generates the COLLOID OSMOTIC HEMOLYSIS- ATP lose permits reduced form of nicotin- amide adenine dinucleotide Ca2+ and Na+ influx, the cell swells and become phosphate (NADPH). NADPH reduces oxidized spheroid rapture glutathione (GSSG). Reduced glutathione (GSH) DISORDERS reduces peroxides and protects proteins, lipids, and ○ Overhydrated stomatocytosis ( Heredity heme iron from oxidation. Hydrocytosis) The methemoglobin reductase pathway converts ○ Dehydrated stomatocytosis ( Heredity ferric heme iron (Fe3+, methemoglobin) to reduced Xerocytosis) ferrous (Fe2+) form, which binds O₂. The Rapoport-Luebering pathway generates 2,3-BPG and enhances O₂ delivery to tissues. The RBC membrane is a lipid bilayer whose hydrophobic components are sequestered from aqueous plasma and cyto- plasm. The membrane provides a semipermeable barrier separating plasma from cytoplasm and maintaining an osmotic differential. RBC membrane phospholipids are asymmetrically distributed. Phosphatidylcholine and sphingomyelin predominate in the outer layer; phosphatidylserine and phosphatidyletha- nolamine form most of the inner layer. Enzymatic plasma to membrane exchange maintains RBC membrane cholesterol.Acanthocytosis and target cells are associated with abnor- malities in the concentration or distribution of membrane cholesterol and phospholipids. RBC transmembrane proteins transport ions, water, and glu- cose and anchor cell membrane receptors Transmembrane Proteins are critical in the ankyrin complex and the actin junctional (4.1) complex to provide the vertical membrane support and to connect the lipid bilayer to the underlying cytoskeleton to MLS 116 - HEMATOLOGY I BSMT-3A maintain membrane integrity and prevent membrane loss. Shape and flexibility of the RBC, which are essential to its function, depend on the cytoskeleton. The cytoskeleton is derived from cytoskeletal (peripheral) proteins on the cyto- plasmic side of the lipid membrane. The major cytoskeletal proteins are a- and B-spectrin heterodimers, which associate in tetramers, and their ends connect to the actin junctional complex forming a hexagonal cytoskeletal lattice adjacent to the cytoplasmic side of the lipid bilayer. Cytoskeletal proteins provide the horizontal or lateral support for the membrane. Hereditary spherocytosis arises from defects in spectrin or proteins forming the ankyrin complex that provide vertical support for the membrane. Hereditary elliptocytosis is due to defects in cytoskeletal proteins that provide horizontal support for the membrane. RBC cytoplasmic K+ concentration is higher than plasma K+, whereas Na and Ca2+ cytoplasmic concentrations are lower. Disequilibria are maintained by membrane enzymes K+-ATPase, Na+-ATPase, and Ca2+-ATPase. Pump failure leads to cation imbalance, water influx, cell swelling, and lysis. Membrane proteins are extracted using sodium dodecyl sulfate, separated using polyacrylamide gel electrophoresis based on their molecular weight and net charge, and stained with Coomassie blue. Glycoproteins are stained with peri- odic acid-Schiff (PAS).