RED BLOOD CELLS by Mary Yvonnette C. Nerves, MD, FPSP Erythropoiesis A process by which early erythroid precursor cells differentiate to become the mature RBCs Primary regulator: ERYTHROPOIETIN - stimulates red cell precursors at all levels of maturation to hasten the maturation process - responsible for stimulating the premature release of reticulocytes into the bloodstream. Erythropoiesis Total erythropoiesis: - total number of red blood cells (RBCs) - measured by the myeloid-erythroid (M:E) ratio from aspirate smears plus the estimate of cellularity from biopsy sections Effective erythropoiesis: - number of viable and functional RBCs available for physiologic needs - reflects the balance between the number of cells produced and their life span - measured by the reticulocyte count, which is normally 1% of the total RBC count Stages of Maturation 1. Pronormoblast (Rubriblast) 2. Basophilic Normoblast (Prorubriblast) 3. Polychromatophilic Normoblast (Rubricyte) 4. Orthochromatic Normoblast (Metarubricyte) 5. Reticulocyte 6. Erythrocyte Pronormoblast Earliest recognizable and largest cell of the erythrocyte series Morphology: - Size: 12 – 20 um - Nucleus: large round, oval, dark violet; fine chromatin; 1 – 2 nucleoli - Cytoplasm: deep blue spotty, basophilic w/ a perinuclear halo - N/C Ratio: 8:1 - BM (%): 1 Basophilic Normoblast Hemoglobin synthesis begins at this stage Morphology: - Size: 10 – 15 um - Nucleus: large round to sl oval; condensed, coarse chromatin; 0 – 1 nucleoli - Cytoplasm: deeply basophilic; clusters of free ribosomes - N/C Ratio: 6:1 - BM (%): 1-4 Polychromatic Normoblast Increased production of hemoglobin pigmentation and decreasing amounts of RNA Last stage in which the cell is capable of mitoses Morphology: - Size: 10 - 15 um - Nucleus: round nucleus, deep staining, may be centrally or eccentrically located; coarse & clumped chromatin - Nucleoli: 0 Morphology: - Cytoplasm: abundant blue-gray (RNA) to pink-gray (hemoglobin) - N/C Ratio: 4:1 - BM (%): 10-20 Orthochromatic Normoblast The last nucleated stage Cannot synthesize DNA and cannot undergo cellular division The NRBC sometimes seen in the peripheral circulation Morphology: - Size: 8 - 10 um - Nucleus: small pyknotic nucleus; dense chromatin; 0 nucleoli - Cytoplasm: abundant red-orange cytoplasm uniform in color - N/C Ratio: 1:2 - BM (%): 5-10 Reticulocyte Slightly larger than the mature RBC with residual amts of RNA Reticulocyte count: an index of bone marrow activity or effective erythropoiesi Morphology: - Size: 8 - 10 um - Nucleus: anucleate cell containing small amt of basophilic reticulum (RNA) - Nucleoli: 0 - Cytoplasm: large amt of blue-pink staining hemoglobin cytoplasm Erythrocyte A biconcave 6 – 8 um disc Life span: 120 days Main function: to transport hemoglobin, a protein that delivers oxygen from the lungs to tissues and cells Contains 90% hemoglobin and 10% H2O normal conc of RBCs varies w/ age, sex & geographic distribution Morphology: - Size: 7 - 8 um - Nucleus: anucleated cell - Nucleoli: 0 - Cytoplasm: pink staining, zone of central pallor is 1/3 of cell diameter devoid of hemoglobin - N/C Ratio: NA Hemoglobin: Structure & Function A conjugated protein that serves as the vehicle for the transportation of O2 and CO2 When fully saturated, each gram of Hgb can hold 1.34 mL of O2 A molecule of Hgb consists of 2 pairs of polypeptide chains (“globin”) and 4 prosthetic heme grps each contg 1 atom of ferrous iron DESCRIPTION of TERMS SIZE DESCRIPTORS Anisocytosis: variation in the sizeof the RBCs due to a pathologic condition Normocytic: normal sized biconcave disc RBC - normal MCV Microcytic: Smaller RBCs less than 6 um - MCV < 80 fl - Defect / Change: abn size due to failure of hgb synthesis - Dse: IDA, Thalassemia, Chronic dse Macrocytic: Larger RBCs greater than 9um - MCV > 90 fl - Defect / Change: impaired DNA synthesis / stress erythropoiesis - Dse: Megaloblastic anemia / liver dse / MDS / Alcoholism / Malaria Macrocytic Microcytic CHROMICITY DESCRIPTORS Normochromic: normal in color; pale central area occupies less than 1/3 - Defect / Change: normal amt of Hgb - Normal indices Hypochromic: an RBC that has a decreased Hgb complement - central pallor exceeds 1/3 of diameter of cell - Defect / Change: reduced Hgb content ( MCHC) - Assoc conditions: IDA / Thalassemia “Hyperchromic”: no central pallor - Defect / Change: greater than normal MCHC - Assoc condition: Spherocytosis “Hyperchromic” Hypochromic Polychromasia: blue-gray coloration - Defect / Change: presence of RNA - Assoc condition: increased erythropoietic activity / hemorrhage / hemolysis SHAPE DESCRIPTORS Poikilocytosis: variation in shape of the RBC - Defect / Change: irreversible alteration of membrane - Assoc conditions: Anemia / Hemolytic states Discocyte: normal biconcave erythrocyte - 6 – 8 um diameter; 0 – 2 um thickness - Aka: Normocyte Normal Red Cells (SEM) Acanthocyte: spheroid w/ 3 – 12 irreg spikes or spicules - Aka: spur cell - decreased cell volume - Defect / Change: inc ratio of chole to lecithin - Assoc conditions: end-stage liver dse Pyruvate kinase def Hemolytic anemia Abetalipoproteinemia Blister cell: contains 1 or more vacuoles - Aka: Bite cells - thinned periphery - Defect / Change: formed by removal of Heinz bodies - Assoc conditions: Hemolytic episodes G6PD def Hemoglobinopathies Codocyte : peripheral rim of Hgb surr by clear area & central hemoglobinized area (bull’s eye) - Aka: target cell - Defect / Change: excess of surface to volume ratio - Assoc conditions: Hemoglobinopathies Thalassemia Liver dse Postsplenectomy Dacryocyte: teardrop or pear-shaped w/ single elongated point or tail - Aka: tear drop cell - Defect / Change: squeezing & fragmentation during splenic passage - Assoc conditions: Myeloid metaplasia Thalassemia Megaloblastic anemia Hypersplenism Drepanocyte: crescent-shaped cell that lacks zone of central pallor - Aka: Sickle cell - Defect / Change: polymerization of deoxygenated Hgb - Assoc conditions: Sickle cell anemia SC disease S-thalassemia Echinocyte: regular 10-30 scalloped short projections evenly distributed / spiny-like - Aka: Burr cell / crenated RBC - Defect / Change: Depletion of ATP Exposure to hypertonic soln Artifact in air drying - Assoc conditions: Uremia Cirrhosis / Hepatitis Chronic renal dse Ovalocyte: egglike or oval-shaped cell - Defect / Change: Hgb has bipolar arrangement Reduction in membrane chole - Assoc conditions: Megaloblastic BM Myelodysplasia Sickle cell anemia Elliptocyte: rod or cigar shape, generally narrower than ovalocytes - Defect / Change: polarization of Hgb - Assoc conditions: Thalassemia Iron def Hereditary elliptocytosis Schistocyte: Fragmented RBCs varying in size & shape - Aka: Helmet cells - Defect / Change: extreme fragmentation produced by damage of RBC by fibrin, altered vessel walls, prosthetic heart valves - Assoc conditions: DIC / TTP / Burns Microangiopathic hemolytic anemia Spherocyte: smaller in diameter than normal RBC w/ concentrated Hgb content; no visible central pallor - Defect / Change: lowest surface area to volume ratio defect of loss of membrane - Assoc conditions: Hereditary spherocytosis Iso- & autoimmune hemolytic anemia Severe burns Hemoglobinopathies Stomatocyte: normal sized cell w/ slitlike area in center - Defect / Change: artifact of slow drying known to have inc permeability to Na+ - Assoc conditions: Hereditary stomatocytosis Acute alcoholism Liver dse RED CELL INCLUSIONS Basophilic Stippling - cytoplasmic remnants of RNA - Fine: thin round dark blue granules uniformly distributed - Defect/Change: represents polychromasia (reticulocyte) - Coarse: medium sized uniformly distributed - Defect/Change: represents impaired erythropoiesis Basophilic Stippling - Assoc conditions: Thalassemia Lead Poisoning Increased reticulocytosis Cabot Ring - rings, loops, or figure eights; red to purple - Defect / Change: remnants of microtubules of mitotic spindle - Assoc conditions: Megaloblastic anemia Dyserythropoiesis Heinz bodies - deep purple irregularly shaped inclusions found on RBC inner surface of membrane - Defect / Change: represent precipitated, denatured Hgb due to oxidative injury - Assoc conditions: Hereditary defects in HMS G6PD def Unstable Hgbs Splenectomized pts Thalassemia Howell-Jolly bodies: coarse round densely stained purple 1-2 um granules eccentrically located on periphery of membrane - Defect / Change: nuclear remnants; contain DNA - Assoc conditions: Megaloblastic anemia Severe hemolytic process Thalassemia Accelerated erythropoiesis Pappenheimer bodies: small, 2-3 um irregular basophilic inclusions that aggregate in small clusters near periphery w/ Wright’s stain - Defect / Change: unused iron (nonheme) deposits - Assoc conditions: Sideroblastic anemia Defective erythropoiesis MDS Hemolytic anemia Thalassemia Ringed Sideroblasts - Nucleated RBC that contains nonheme iron particles (siderotic granules) arranged in ring form - Defect / Change: excessive iron overload in mitochondria of normoblasts - due to defective heme synthesis - Assoc conditions: Sideroblastic anemia MDS Ringed Sideroblasts Prussian blue iron stain showing excess accumulation of iron as ferritin in mitochondria ringing nucleus. Siderocyte: non-nucleated cell containing iron granules - Defect / Change: excessive iron overload in mitochondria of normoblasts - due to defective heme synthesis - Assoc conditions: Sideroblastic anemia MDS Autoagglutination: clumping of RBCs - Defect / Change: presence of antibody - Assoc conditions: Cold agglutinin AHA Rouleaux Formation: alignment of RBCs linear appearing as stacks of coins - Defect / Change: concentration of fibrinogen & immunoglobulin - Assoc conditions: MM / Waldenstrom’s macroglobulinemia Red Cell Studies Hematologic tests used to measure several important parameters that reflect rbc structure and function: 1) Hemoglobin determination 2) Erythrocyte count 3) Hematocrit 4) Erythrocyte Indices: MCH, MCHC, MCV 5) Reticulocyte Count 6) Osmotic Fragility Test 7) Erythrocyte Sedimentation Rate (ESR) Adult Reference Ranges for Red Blood Cells Measurement (units) Men Women 13.6-17.2 12.015.0 Hematocrit (%) 39-49 33-43 Red cell count (106/μL) 4.3-5.9 3.5-5.0 Hemoglobin (gm/dL) Reticulocyte count (%) 0.5-1.5 Mean cell volume (μm3) 82-96 Mean corpuscular hemoglobin (pg) 27-33 Mean corpuscular hemoglobin concentration (gm/dL) 33-37 RBC distribution width 11.5-14.5 Hemoglobin - involves lysing the erythrocytes, thus producing an evenly distributed solution of hemoglobin in the sample - Hemiglobincyanide Mtd: blood is diluted in a soln of K3Fe(CN6). The K3Fe(CN6) oxidizes Hgbs to hemiglobin (metHgb) and K cyanide provides cyanide ions to form HiCN, w/c has a broad absorption max at a wl of 540 nm Erythrocyte Count - involves counting the number of rbcs per unit volume of whole blood. - expressed as number of cells per unit volume, specifically cells/µL - NV: Female = 4.2 - 5.4 x 106/µL Males = 4.7 - 6.1 x 106/µL Hematocrit - sometimes referred to as the Packed Cell Volume (PCV) or volume of packed red cells - is the ratio of the volume of RBCs to that of the whole blood - varies with age and sex - expressed as a percentage or as a decimal fraction Plasma Buffy coat Red cells Erythrocyte Indices 1) Mean Cell Volume (MCV) - average volume of red cells - calculated from the Hct and RBC count MCV = Hct RBC (in millions/uL) x 1000 - expressed in femtoliters (fl) or cubic micrometers 2) Mean Cell Hemoglobin (MCH) - content (weight) of Hgb of the average red cell - calculated from the Hgb and RBC count MCH = Hgb (in g/L) RBC (/L) - value is expressed in picograms (pg) 3) Mean Cell Hemoglobin Concentration (MCHC) - the average conc of Hgb in a given volume of packed red cells - calculated from the Hgb conc & the Hct MCHC = Hgb (in g/dL) Hct - expressed in g/dL Morphologic Classification of Anemias Type of Anemia Blood Constants MCV (mm3 or fl) MCHC (g.Hb/dl.RBC or mmol/l) 60-87 20-30 Macrocytic normochromic 103-160 32-36 Normocytic normochromic 87-103 32-36 Microcytic normochromic 60-87 32-36 Microcytic hypochromic Reticulocyte Count - Principle: Reticulocytes are immature nonnucleated red cells that contain RNA and continue to synthesize Hgb after the loss of the nucleus - Supravital staining: blood is briefly incubated in a soln of new MB or BCB, the RNA is precipitated as a dye-ribonucleoprotein complex dark blue network (reticulum or filamentous strand) - NV: 0.5 – 1.5% or 24 – 84 x 109/L Osmotic Fragility Test (OFT) - a measure of the ability of red cells to take up fluid without lysing - Red cells are suspended in a series of tubes contg hypotonic solns of NaCl solns varying from 0.9% to 0.0%, incubated at room temp for 30 mins and centrifuged - the percent hemolysis in the supernatant solns is measured & plotted for each NaCl conc. - The larger the amount of red cell membrane (surface area) in relation to the size of the cell, the more fluid the cell is capable of absorbing before rupturing - Cells that are more spherical, w/ a decreased surface/volume ratio, have a limited capacity to expand in hypotonic solns & lyse at a higher conc of NaCl than do normal biconcave cells OFT - Cells that are hypochromic & flatter have a greater capacity to expand in hypotonic solns, lyse at a lower conc than do normal cells, & are said to have decreased osmotic fragility - Cells with increased surface/volume ratio are osmotic resistant IDA, thalassemia, liver dse, & reticulocytosis Erythrocyte Sedimentation Rate (ESR) - detect and monitor an inflammatory response to tissue injury (an acute phase response) in which there is a change in the plasma conc of several proteins - Principle: When well-mixed venous blood is placed in a vertical tube, RBCs will tend to fall toward the bottom. The length of the fall of the top of the column of RBCs in a given interval of time is called the ESR - ESR is affected by (3) FACTORS: a) erythrocytes b) plasma composition c) mechanical / technical factors - Red Cell Factors: Anemia increases ESR (change in RBC plasma ratio favors rouleaux fotn) ESR is directly proportional to the weight of the cell aggregate & inversely proportional to the surface area Microcytes sediment slower than macrocytes Rouleaux accelerate the ESR Red cells w/an abnormal or irregular shape hinder rouleaux fotn & lower the ESR - Plasma Factors: Elevated levels of fibrinogen accelerate ESR Albumin & lecithin retard ESR Cholesterol accelerate ESR - Mechanical / Technical Factors: A tilt of 3o can cause errors up to 30% ESR ESR increases as the temp increases ESR tubes with a narrower than standard bore will generally yield lower ESR ESR stands fro > 60 mins falsely elevated ESR Greater conc of EDTA falsely low ESR - Methods: Westergren Mtd / Wintrobe Mtd ERYTHROCYTE DISORDERS Two main disorders affecting RBCs: 1. Polycythemia (Erythrocytosis) - an elevated Hct level above the normal range 2. Anemia - a reduction below normal limits of the total circulating red cell mass Pathophysiologic Classification of Polycythemia Relative Reduced plasma volume (hemoconcentration) Absolute Primary Polycythemia vera, rare erythropoietin receptor mutations (low erythropoietin) Secondary High erythropoietin Appropriate: lung disease, high-altitude living, cyanotic heart disease Inappropriate: erythropoietin-secreting tumors (e.g., renal cell carcinoma, hepatocellular carcinoma, cerebellar hemangioblastoma) POLYCYTHEMIA May be classified into (2) major conditions: 1) Relative Polycythemia - an increase in the Hct or red cell count as a result of decreased plasma volume - total red cell mass is NOT increased - Assoc conditions: acute dehydration or hemoconcentration / pts on diuretic therapy / Gaisbock’s syndrome (psedopolycythemia or stress erythrocytosis) - BM: Normal 2) Absolute (or Secondary) Polycythemia - an erythropoietin mediated increase in RBCs and Hgb due primarily to a hypoxic situation - increase in the total red cell mass in the body assoc w/ normal or sl increased plasma volume - Assoc conditions: tumors / anabolic steroids / & renal dso such as cystic dse, hydronephrosis / & adrenal cortical hyperplasia - BM: Erythroid hyperplasia 3) Polycythemia rubra vera (Primary Erythrocytosis) - an absolute increase in all cell types, RBCs, WBCs and platelets - not dependent on erythropoietin levels - BM: all three cell lines increased (panhyperplasia) ANEMIA Decreased oxygen carrying capacity of the blood Anemia may also be "defined" in terms of the Hb content Hb < 12 g/dL in an adult male Hb < 11 g/dL in an adult female Anemia Males: Hb < 13.5 Hct < 41 Female: Hb < 12 Hct < 36 MCV Microcytic MCV < 80 Iron deficiency anemia Thalassemia Anemia of Chronic disease Sideroblastic anemia Normocytic MCV 80-100 Macrocytic MCV > 100 Reticulocyte Count Megaloblastic anemia Alcoholic liver disease Low Marrow Failure Aplastic anemia Myelofibrosis Leukemia /Metastasis Renal failure Anemia of Chronic disease High Sickle cell anemia G6PD def anemia Hereditary spherocytosis AIHA PNH ANEMIAS SECONDARY TO BLOOD LOSS Acute: e.g., hemorrhage due to trauma, massive GI bleeding, or child delivery. Usually the iron stores remain normal. Chronic: e.g., bleeding peptic ulcer or excessive menstrual bleeding. HYPOPROLIFERATIVE ANEMIAS (Impaired Production) reduced production of red cells can be subdivided into: deficiency of haematinics iron deficiency B12 and folate deficiency dyserythropoiesis (production of defective cells) anaemia of chronic disorders (AOCD) myelodysplasia sideroblastic anaemia marrow infiltration (myelophthisic anemia) aplasia (failure of production of cells) aplastic anaemia red cell aplasia Iron Deficiency Anemia Normal forms of iron (Fe) and iron metabolism Functional iron is found in Hb, myoglobin, and enzymes (catalase & cytochromes) Ferritin: physiological storage form Hemosiderin: degraded ferritin + lysosomal debris (Prussian blue positive) Iron is transported by transferrin causes: Dietary deficiency: elderly, children and poor Increased demand: children & pregnant women Decreased absoprtion: generalized malabsorption after gastrectomy Chronic blood loss: GI bleeding (e.g. peptic ulceration, carcinoma of stomach or colon) menorrhagia urinary tract bleeding Hook worm (Ancylostoma duodenale adult worm sucks 0.2 ml blood/day) Lab Findings: Microcytic, hypochromic anemia. Low serum iron BM: show absence of iron Ferritin: Low serum ferritin indicates low body stores of iron Transferrin: These carrier proteins will be unsaturated and available to bind iron, hence the Total Iron Binding Capacity (TIBC) is increased with anemia. Anemia of Chronic Disease (AOCD) Char by iron being trapped in BM macrophages Can be grouped in 3 categories: - chronic microbial infections (eg. Osteomyelitis) - chronic immune disorders (eg. RA) - Neoplasms (eg. lymphoma, breast/lung CA) Chronic inflamm dso inc IL-1, TNF, IF-Gamma - reduction in renal erythropoietin marrow erythroid precursors do notproliferate - hepcidin synthesis in liver inhibits release of iron Labs: low serum iron increased serum ferritin decreased TIBC normochromic, normocytic anemia or hypochromic, microcytic anemia Megaloblastic Anemia A group of dso in which the blood and BM hematopoietic cells display changes Pathogenesis: impaired DNA synthesis (delayed mitoses) while RNA is not impaired; this produces nuclear-cytoplasmic asynchrony Megaloblastic anemias can be divided into groups: - anemia caused by B12 deficiency - anemia caused by folate deficiency - anemias nonresponsive to either therapy important background knowledge: B12: vitamin B12 is required for DNA replication and inhibition of transcription of DNA to RNA B12 is normally absorbed from gut by the following mechanism: - secretion of intrinsic factor by parietal cells in stomach - binding of intrinsic factor and vitamin B12 in lumen - intrinsic factor- B12 complex is absorbed in terminal ileum through pinocytotic vesicles Folate: - folate is required for DNA replication and inhibition of transcription of DNA to RNA lack of B12 or folate means that RNA builds up and the cells become too large Causes: causes of vitamin B12 deficiency (pernicious anaemia) lack of intrinsic factor - atrophic gastritis - parietal cells are destroyed gastrectomy malabsorption of B12 not related to lack of intrinsic factor - tropical sprue or bacterial overgrowth of terminal ileum - ileal disease (e.g. Crohn's disease affecting the terminal ileum) - fish tape-worm (these attach to intestinal wall, and therefore in large enough numbers, may prevent B12intrinsic factor complex absorption in terminal ileum) - poor diet - rare causes of folate deficiency poor diet - especially in alcoholics malabsorption - coeliac disease increased cell turnover (e.g. leukaemia, chronic haemolysis, pregnancy) antifolate drugs (e.g. phenytoin) Peripheral Blood Findings: WBCs Normal or decreased RBCs Decreased Hgb Decreased MCV Increased (usualy > 100 fl) MCH Increased MCHC Normalor slightly decreased Platelet count Normal or decreased Reticulocyte count Decreased Reticulocyte Count Normal or decreased Macrocyte normal, no hyersegmented neutrophils Macro-ovalocytes, Hypersegmented neutrophils BM not megaloblastic R/O Refractory anemia, Sideroblastic anemia, Myelodysplasia, drug induction, liver disease, Aplastic anemia Increased Hemolytic Hemorrhagic deficiency Morphologic Abnormalities: Large RBC's with nuclear-cytoplasmic dyssynchrony Ovalocytes: The large RBC's tend to have an oval-shape. Hypersegmented Neutrophils: One of the earliest signs of disease. 5 or 6 lobes Howell-Jolly Bodies: Nuclear fragments seen in Megaloblastic anemia. Aplastic Anemia pancytopenia associated w/ a severe reduction in the amt of hematopoietic tissue that results in deficient production of blood cells Etiology: Acquired idiopathic Chemical agents Physical agents Viral infections Inherited Fanconi’s anemia Pure red cell aplasia: erythrocyte stem cells are suppressed, but the other formed elements of blood are unaffected Anemia due to isolated depletion of erythroid precursors in the marrow, and may be acute or chronic. Lab Findings: - Normochromic, normocytic or macrocytic anemia - Reticulocytes are decreased or absent because it is hypoproliferative. - BM: hypocellular or dry tap reduction in all cell lines HEMOLYTIC ANEMIAS (Increased Destruction) Grp of dso that can be inherited, acquired,or drug-induced Char by an increased red cell destruction or shortened survival of the RBC Char by increased BM activity, polychromasia, nucleated RBCs and an increased reticulocyte count w/ stress reticulocytes Hemolytic anemias share the ff. features: 1. shortened red cell life span, that is, premature destruction of red cells 2. elevated erythropoietin levels and increased erythropoiesis in the marrow & other sites 3. accumulation of products of Hgb catabolism, due to an increased rate of red cell destruction HEMOLYTIC ANEMIAS Acquired Immune-mediated - Autoimmune - Alloimmune (Transfusion) - Drug-induced Microangiopathic Infection Hereditary Enzymopathies Membranopathies Hemoglobinopathies INTRINSIC DEFECTS Hereditary Spherocytosis abnormal cell membrane assoc cytoskeleton causing red cells to be spherical and fragile principle defect is an abnormality of the membrane protein ankyrin Lab findings: Normocytic, hyperchromic anemia (normal MCV and increased MCHC) - increased pigment catabolism, erythroid hyperplasia, & reticulocytosis - red cells with increased OFT Glucose-6-Phosphate Dehydrogenase Deficiency Normal: G6PD metabolises glucose, and forms small amounts of ATP (which maintains the cell cytoskeleton and membrane) and NADPH (which mops up free radicals) G6PD def renders the cell susceptible to damage by free radicals an X-linked recessive condition, in which haemolytic crises are precipitated by infections or certain drugs Lab findings: poikilocytes & spherocytes, & Heinz bodies (stain w/ methyl violet) HEMOGLOBINOPATHIES Normal Hgb: HbA / HbF / HbA2 (adult) Hb Gower-1 and 2 / Hb-Portland (embryonic) Hemoglobin-A alpha2, beta2 Predominant hemoglobin in adults Hemoglobin-A2 alpha2, delta2 Found in normal adults Hemoglobin-F alpha2, gamma2 Fetal (cord) hemoglobin, with higher O2-binding affinity Hemoglobin-S alpha2, beta2 Sickle-Cell Hemoglobin. Sickle crisis (beta: Glu-6 can result from low O2-tension. --> Val) Hemoglobin-C alpha2, beta2 Hemoglobin-C Disease. Second most (beta: Glu-6 common hemoglobinopathy. --> Lys) THALASSEMIA Caused by impaired production of one of the polypeptide chains of the Hb molecule Epidemiology: Mediterranean, African & Asian ancestry autosomal recessive disease Types according to clinical severity: thalassaemia major = homozygote; thalassaemia minor = heterozygote Types according to molecular defect: beta thalassaemia alpha thalassaemia Beta-Thalassemia Major (Homozygous state): - severe hypochromic, microcytic anaemia, hepatosplenomegaly, marrow hyperplasia causing skeletal deformities, haemochromatosis develops with repeated transfusions Minor (Heterozygous states): - reduction in HbA, but increase in HbA2; mild anaemia with hypochromia Clinical Nomenclature Genotype Disease Molecular Genetics β-Thalassemias Thalassemia major Homozygous β0thalassemia (β0/β0) Severe; requires blood transfusions Homozygous β+thalassemia (β+/β+) Thalassemia intermedia β0/β β+/β+ Severe, but does not require regular blood transfusions Thalassemia minor β0/β β+/β Asymptomatic with mild or absent anemia; red cell abnormalities seen Rare gene deletions in β0/β0 Defects in transcription, processing, or translation of β-globin mRNA Alpha-Thalassemia note that there are 4 copies of the alpha globin gene (not 2), and therefore four possible degrees of alpha thalassaemia exist 3 good copies - silent carrier 2 good copies - mild anaemia with microcytosis 1 good copy - moderate haemolytic anaemia with hypochromia and mycrocytosis; HbH (tetramer of beta) 0 good copies - lethal in utero (hydrops fetalis) α-Thalassemias Hydrops fetails -/--/- Lethal in utero without transfusions HbH disease -/--/α Severe; resembles βthalassemia intermedia α-Thalassemia trait -/-α/α (Asian) -/α-/α (black African) Asymptomatic, like βthalassemia minor Silent carrier -/αα/α Asymptomatic; no red cell abnormality Mainly gene deletions Thalassemia Major Patient with thalassemia major due to heterozygous hemoglobin E/B thalassemia. Note prominent target cells, anisopoikilocytosis, and three nucleated red cells (normoblasts) Sickle Cell Disease Endemic to Sub-saharan Africa, due to heterozygous advantage conferred against Falciparum Malaria (infected RBC's preferentially sickle and are thus taken to the spleen and sequestered, limiting the spread of infection) PATHOGENESIS: Point-mutation of Glu Val at 6th position of beta-globin chain Pathophysio: abn Hgb polymerises at low O2 saturation causing abnormal rigidity and deformity of red cells and become abnormality fragile (and undergo haemolysis and sludge in small vessels) autosomal recessive, with a point mutation in beta gene forming an abnormal HbS; more common in Negroes Lab. Findings Smear: normochromic, normocytic anemia, increased polychromasia, normoblasts are present, numerous target cells, Howell-Jolly and Pappenheimer bodies are present, sickle cells OFT decreased BM: normoblastic hyperplasia w/ increased iron storage Electrophoresis: no HbA, 80% HbS (SCD) Sickle Cells (SEM) Scanning electron micrograph (SEM) showing sickle cells obstructing small vessel. EXTRINSIC DEFECTS Immune Hemolytic Anemias Dso in w/c erythrocyte survival is reduced because of the deposition of Ig &/or ` complement on the red cell membrane Classification: 1. Autoimmune Hemolytic Anemia 2. Isoimmune Hemolytic Anemia 3. Drug-induced Hemolytic Anemia LAB: (+) direct & indirect antiglobulin tests Agglutination of erythrocytes is seen on this peripheral blood smear Coomb’s Test Traumatic Hemolytic Anemia Char by striking morphologic abn of the red cells, w/c include fragments (schistocytes) & irregularly contracted cells (triangular cells, helmet cells) MICROANGIOPATHIC HEMOLYSIS: RBC's being damaged by intravascular fibrin-clots, in small vessels. DIC, TTP, HUS. MACROANGIOPATHIC HEMOLYSIS: Damage by artifical heart valves. Have a nice day!