4Definition In addition to the death of aging (senescent) RBCs, there is age-independent RBC destruction (random hemolysis) in normal subjects in the range of less than 0.05 to 0.5 percent per day.This latter process is appreciably increased in hemolytic states.
5September 9, :00 amDefinitionAnemia, however, is not invariably a consequence of hemolysis as the bone marrow can increase erythropoiesis to compensate for the increased destruction.Under maximal stimulation, the normal marrow is capable of undergoing hyperplasia until its production rate increases about six to eight times.
6September 9, :00 amDefinitionWith optimal marrow compensation, the survival of red cells in the circulation can theoretically decrease from the normal 120 days to as few as days without anemia developing.Such an increase in both destruction and production of RBC can result in a compensated hemolytic state without anemia being present, so-called compensated hemolytic disease.
7September 9, :00 amDefinitionWhen red cell survival is so short that anemia develops despite a vigorous erythropoietic response,the term hemolytic anemia is appropriate.
8September 9, :00 amPathophysiology:There are two mechanisms of hemolysis: 1.Intravascular hemolysis: is the destruction of red blood cells in the circulation with the release of cell contents into the plasma. 2.Extravascular hemolysis: is the removal and destruction of red blood cells with membrane alterations by the macrophages of the spleen and liver.
9HEMOLYTIC ANEMIA Anemia of increased destruction: September 9, :00 amHEMOLYTIC ANEMIAAnemia of increased destruction:Normochromic,normocytic anemia.Shortened RBC survival.Reticulocytosis - Response to increased RBC destruction.Increased indirect bilirubin.Increased LDH.
11ReticulocytesUnder steady state conditions, the rate of RBC production equals the rate of RBC loss.In survival of mature RBC of 100 days, 1 percent of RBCs are removed from the circulation each day.To achieve a constant RBC mass, RBC losses must be replaced with an equal number of reticulocytes during the same time period.
12ReticulocytesReticulocytes normally survive in the circulation for one day; after this time they lose their reticulum (RNA) and become mature red blood cells.Under steady-state conditions reticulocytes will represent approximately 1 percent of total circulating RBC .Since the normal RBC count is approximately 5 million/microL, the bone marrow must produce approximately 50,000 reticulocytes/microL of whole blood each day in order to achieve a stable RBC mass.Lesser rates of RBC production, if persistent, lead to anemia.
13ReticulocytesA normal bone marrow replete with iron, folate, and cobalamin can increase erythropoiesis in response to EPO about 5-fold in adults and 7- to 8-fold in children.Thus, under optimal conditions, steady-state absolute reticulocyte counts as high as 250,000/microL are possible in the adult.
14Reticulocyte Count Relative reticulocyte count September 9, :00 amReticulocyte CountRelative reticulocyte count% of all RBC (normal %)Absolute reticulocyte countRelative reticulocyte count x RBC countNormal 50,000-75,000/µlExamples:1.1% x 4.96 x106 = 55,000/ml12.2% x 2.05 x106 = 250,000/ml
17Corrected reticulocyte count = reticulocyte count (patient hematocrit / 45) Since the reticulocyte count represents a ratio (number of reticulocytes divided by the total number of erythrocytes), a decrease in the total RBC count may result in an increase in the reticulocyte count even if reticulocyte production by the marrow is not increased.In order to correct for this, the reticulocyte count is multiplied by the patient’s hematocrit divided by a normal hematocrit (45%):
18RPI = corrected reticulocyte count / correction factor Under severe erythropoietin stimulus, reticulocytes may be released from the bone marrow prematurely and must finish their maturation in the peripheral blood (shift reticulocytes).Since these prematurely released cells exist as reticulocytes for more than 1 day, they are, in effect, “counted” more than once in the reticulocyte count (remember: the normal reticulocyte count presumes that the cells exist as reticulocytes for only 1 day, rather than 2 or more days).In order to correct for these shift reticulocytes, the corrected reticulocyte count has to be divided by a correction factor, giving the RPI.
25HEMOLYTIC ANEMIAMature RBC has lost its nucleus, mitochondria, and RNA and is left with three major components:Hemoglobin, the major protein of the red cell.The RBC membrane, the surface area of which must be about 40 percent greater than that of a sphere of identical volume to allow the RBC to undergo elliptical deformation in the microvasculature.Membrane proteins need to allow for such deformation and elastic recoil.In addition, ion pumps and channels regulate RBC volume via the entry and content of water and the cations sodium, potassium, calcium, and magnesium.
26HEMOLYTIC ANEMIAMetabolic machinery to generate ATP needed by the cation pumps, 2,3 DPG that allows hemoglobin to take up and release oxygen, and reducing power (eg, glutathione, NADPH) to protect the oxygen-rich RBC from oxidant injury.
27HEMOLYTIC ANEMIAHemoglobin molecules unstable to oxidant challenge, as in hemoglobin Köln.Membrane injury due to the production of excess alpha or beta globin chains.Unstable or missing RBC membrane proteins, as in hereditary (congenital) spherocytosis and paroxysmal nocturnal hemoglobinuria.Abnormalities of RBC hydration, as in xerocytosis.Defective glycolysis, as in pyruvate kinase deficiency.Abnormalities in availability of reducing power (NADPH), rendering the RBC susceptible to oxidant challenge, as in glucose-6-phosphate dehydrogenase (G6PD) deficiency.
30The red cell membrane consists of three main components: • A phospholipid bilayer• Various integral membrane proteins and glycoproteins embedded in the phospholipid bilayer, some of which extend through the membrane into the cytoplasm (transmembrane proteins). Two of the most important of these cell transmembrane proteins are the band 3 protein, an important anion transport protein, and glycophorin, which carries red cell antigens. Both of these proteins also serve as anchoring sites for cytoskeleton proteins.• A cytoskeleton scaffold, which gives the red cell its characteristic shape.The cytoskeleton is composed predominantly of spectrin. A protein called ankyrin is required to bind spectrin to the band 3 protein.
32PathophysiologyDefective vertical attachment between the phospholipid bilayer and the cytoskeleton scaffold.Loss of phospholipids from the cell membrane. Consequently, the surface area of the RBC decreases, and the cell gradually assumes the shape of a sphere (the shape with the highest volume to surface area ratio).Spherocytic RBCs are less flexible than normal (biconcave disk) RBCs, and, consequently, they are selectively trapped and destroyed in the spleen.The spleen also plays an important role in the loss of cell membranes by the RBCs (a process known as splenic conditioning).
33Hereditary Spherocytosis Most cases (approximately three-fourths) are inherited in an autosomal dominant fashion.The remainder of cases are inherited as autosomal recessive, which are often more severe. A significant number of patients have no family history of the illness and probably represent new mutations.The prevalence of HS in western countries has been estimated as 1 in approximately 5,000.
35Clinical Manifestations Highly variable, from asymptomatic without anemia to severe chronic hemolysis.Mild or moderate anemia, and hyperbilirubinemia (which may be intermittent) and mild splenomegaly.Bilirubin gallstones are common.Like other patients with chronic hemolytic anemias, patients with HS may have exacerbation of anemia associated with infections, aplastic crises due to parvovirus B19 infection,Megaloblastic anemia associated with folate deficiency.Patients with autosomal recessive HS may have severe anemia, with hemoglobin as low as 4 to 6 g/dL.
37Diagnosis of Hereditary Spherocytosis MCV that is normal to slightly low, with an increased MCHC.The blood smear shows microspherocytes .The reticulocyte count is increased (~5–20%).A direct antiglobulin (Coombs’) test should be done to exclude an immune hemolytic anemia.
39Osmotic fragility test The classic laboratory test for HS is the osmotic fragility test.In this test, erythrocytes are incubated in saline solutions with osmolality ranging from normal to pure water.Percent hemolysis is measured by spectrophotometry.Erythrocytes from patients with HS hemolyze at higher saline concentrations than normal cells.The difference is exaggerated by incubating the cells for 24 hours in the absence of glucose.It is important to remember that increased osmotic fragility is not specific for HS; any cause of spherocytosis results in increased osmotic fragility
43Hereditary Spherocytosis September 9, :00 amHereditary SpherocytosisAutosomal dominant disorderAbnormality in RBC membrane proteinClinical and laboratory findingsSplenomegalyChronic hemolytic anemiaSpherocytes on peripheral smearTreatmentSplenectomy
44Hereditary Elliptocytosis Autosomal dominantDefective horizontal stability in the cytoskeleton.Most cases are caused by mutations in alfa spectrin, resulting in impaired assembly of the alfa /beta spectrin tetramers.The cytoskeleton is less rigid and mor easily deformed.Neonatal hyperbilirubinemia is common.Most cases of HE in the United States are asymptomatic, with normal RBC survival and no anemia.Examination of a peripheral blood smear is the key diagnostic test .The presence of more than 15% elliptocytes suggests HE.Splenectomy is not curative but may be beneficial in the rare severe cases that require therapy.
46HEMOLYTIC ANEMIA Membrane abnormalities - Enzymopathies September 9, :00 amHEMOLYTIC ANEMIA Membrane abnormalities - EnzymopathiesDeficiencies in Hexose Monophosphate ShuntGlucose 6-Phosphate Dehydrogenase DeficiencyDeficiencies in the EM PathwayPyruvate Kinase Deficiency
47Glucose 6-Phosphate Dehydrogenase Functions Glucose-6-Phosphate Dehydrogenase Deficiency is the first enzyme in the hexose monophosphate shunt, which is required to generate the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH).NADPH is required for the regeneration of glutathione by the enzyme glutathione reductase.
48Glucose 6-Phosphate Dehydrogenase deficiency In the absence of sufficient glutathione, hemoglobin is oxidized and precipitates in the cell.This damages the cell, resulting in hemolysis.The aggregates of oxidized hemoglobin are plucked out of the cell by the spleen, resulting in characteristic “bite” or “blister” cells.
50Glucose 6-Phosphate Dehydrogenase deficiency The level of G6PD is highest in reticulocytes and declines with increasing age of the red cell.In normal cells, the half-life of the enzyme is approximately 62 days.The activity level of the enzyme remains sufficient to protect against oxidative stress even in older cells.In patients with G6PD deficiency, the enzyme level declines faster, so older cells are not protected against oxidative stress.
51Glucose 6-Phosphate Dehydrogenase Different Isozymes September 9, :00 amGlucose 6-Phosphate Dehydrogenase Different IsozymesLevel needed for protection vs ordinary oxidative stress
52Glucose 6-Phosphate Dehydrogenase variants Class I variants: Severe enzyme deficiency (<10% of normal) andchronic hemolysisClass II variants: Severe enzyme deficiency, but intermittent instead of chronic hemolysis(G6PD Mediterranean).Class III variants: Mild to moderate enzyme deficiency (10–60% ofnormal), with intermittent hemolysis usually precipitated by infection or oxidative drugs or chemicals(African variant).Class IV variants: Normal enzyme activity and no anemia or hemolysisClass V variants: Increased enzyme activity, without anemia or hemolysis
53Glucose 6-Phosphate Dehydrogenase Functions September 9, :00 amGlucose 6-Phosphate Dehydrogenase FunctionsRegenerates NADPH, allowing regeneration of glutathioneProtects against oxidative stressLack of G6PD leads to hemolysis during oxidative stressInfectionMedicationsFava beansOxidative stress leads to Heinz body formation, extravascular hemolysis.
54Glucose-6-Phosphate Dehydrogenase Deficiency September 9, :00 amGlucose-6-Phosphate Dehydrogenase DeficiencySex-linked disorderEffects > 200 million peopleHemolytic anemia occurs in the presence of stress (infection or drugs)African form - mild hemolysisMediterranean form - more severeUnique sensitivity to fava beans
55the normal G6PD is designated as type B. About 20% of individuals of African descent have a G6PD (designated A+) that differs by a single amino acid and is electrophoretically distinguishable but functionally normal.Among the clinically significant G6PD variants, the most common, the so-called A- type, is due to two base substitutions and is encountered primarily in individuals of central African descent. The A- G6PD has the same electrophoretic mobility as the A+ type, but it is unstable and has abnormal kinetic properties. This variant is found in about 11% of African-American males.A second relatively common G6PD variant is encountered among groups of Mediterranean origin, particularly Sardinians and Sephardic Jews; this variant is more severe than the A- variant and may result in nonspherocytic hemolytic anemia in the absence of known oxidative stress.
56Diagnosis of Glucose-6-Phosphate Dehydrogenase Deficiency There is an easy fluorescent screening test for NADPH production from glucose-6- phosphate and NADP.If this test is abnormal, a quantitative assay can be performed to confirm the diagnosis and assess the severity of the deficiency.
57Diagnosis of Glucose-6-Phosphate Dehydrogenase Deficiency The G6PD level can be normal in a patient with G6PD deficiency shortly after a hemolytic episode, giving a false-negative result.This occurs if the reticulocyte count is high since reticulocytes have higher G6PD levels than older cells.If you get a normal G6PD level in a patient who you really think has G6PD deficiency, repeat the laboratory test in a few weeks when cells of all ages are again present.
59Treatment of Glucose-6-Phosphate Dehydrogenase Deficiency The main treatment for G6PD deficiency is to avoid conditions that predispose to hemolysis.Patients with G6PD deficiency should not be given medications that cause hemolysis; they should be advised to avoid exposure to naphthalene (mothballs), fava beans, and other hemolytic agents.Infections should be treated promptly.Infants with marked hyperbilirubinemia may require exchange transfusions.Women known to be heterozygous for G6PD deficiency should avoid oxidative drugs while pregnant and nursing to prevent inducing hemolytic episodes in a susceptible fetus or infant.
61Pyruvate Kinase Deficiency The most common enzyme deficiency in the Embden-Meyerhof (glycolytic) pathway .However, it is far less common than G6PD deficiencyAutosomal recessive.Neonatal hyperbilirubinemia, Older children and adults have chronic hemolysis, Splenomegaly is common.Infections, surgery, and pregnancy may precipitate acute exacerbations of hemolysis.Aplastic crises may occur due to infection with parvovirus B19.
62PathophysiologyErythrocytes with PK deficiency generate less adenosine triphosphate (ATP) and NADH from glucose.There is decreased Na+,K+-ATPase activity, with consequent cellular dehydration.The exact mechanism of hemolysis is unknown, but it is thought that there are abnormalities in membrane function.2,3-Diphosphoglycerate (DPG) accumulates in RBCs; since increased 2,3-DPG facilitates O2 unloading, patients tolerate anemia well and are relatively asymptomatic despite decreased blood hemoglobin.
63Diagnosis of Pyruvate Kinase Deficiency A variety of enzyme assays are available. It may be difficult to demonstrate decreased enzyme activity if cells with the most severe deficiency are largely destroyed and cells with higher enzyme levels are relatively preserved.It may be helpful to study relatives, although there is overlap in enzyme levels between heterozygotes and the normal population.
64Treatment of Pyruvate Kinase Deficiency Patients with more severe hemolysis may require transfusion support.Splenectomy can be beneficial in severe cases; it results in striking reticulocytosis (up to 50–60%).
67EXTRACORPUSCULAR HEMOLYSIS Nonimmune September 9, :00 amEXTRACORPUSCULAR HEMOLYSIS NonimmuneMechanical: Trauma secondary to high velocity jets (malfunctioning prosthetic aortic valves) or fibrin stands across vessels that shear RBCs in disseminated intravascular coagulation and thrombotic thrombocytopenic purpura-hemolytic uremic syndrome.Infectious: malaria and babesiosis.Thermal burn.Osmotic: hypotonic solutions(dialysis fluid or infused intravenously, urologic procedures, freshwater near-drowning).Snake and insect bites.
72HEMOLYTIC ANEMIA - IMMUNE September 9, :00 amHEMOLYTIC ANEMIA - IMMUNEDrug-Related HemolysisAlloimmune HemolysisHemolytic Transfusion ReactionHemolytic Disease of the NewbornAutoimmune HemolysisWarm autoimmune hemolysisCold autoimmune hemolysis
73IMMUNE HEMOLYTIC ANEMIA General Principles September 9, :00 amIMMUNE HEMOLYTIC ANEMIA General PrinciplesAll require antigen-antibody reactionsTypes of reactions dependent on:Class of AntibodyNumber & Spacing of antigenic sites on cellAvailability of complementEnvironmental TemperatureFunctional status of reticuloendothelial systemManifestationsIntravascular hemolysisExtravascular hemolysis
74IMMUNE HEMOLYTIC ANEMIA General Principles - 2 September 9, :00 amIMMUNE HEMOLYTIC ANEMIA General Principles - 2Antibodies combine with RBC, & eitherActivate complement cascade, &/orOpsonize RBC for immune systemIf 1, if all of complement cascade is fixed to red cell, intravascular cell lysis occurs.If 2, &/or if complement is only partially fixed, macrophages recognize Fc receptor of Ig &/or C3b of complement &phagocytize RBC, causing extravascular RBC destruction.
76IMMUNE HEMOLYTIC ANEMIA Coombs Test - Direct September 9, :00 amIMMUNE HEMOLYTIC ANEMIA Coombs Test - DirectLooks for immunoglobulin &/or complement of surface of red blood cell (normally neither found on RBC surface)Coombs reagent ( combination of anti-human immunoglobulin & anti-human complement Mixed with patient’s red cells);if immunoglobulin or complement are on surface, Coombs reagent will link cells together and cause agglutination of RBCs.
77IMMUNE HEMOLYTIC ANEMIA Coombs Test - Indirect September 9, :00 amIMMUNE HEMOLYTIC ANEMIA Coombs Test - IndirectLooks for anti-red blood cell antibodies in the patient’s serum, using a panel of red cells with known surface antigens.Combine patient’s serum with cells from a panel of RBC’s with known antigens.Add Coombs’ reagent to this mixture If anti-RBC antigens are in serum, agglutination occurs.
81AUTOIMMUNE HEMOLYSIS Warm Type September 9, :00 amAUTOIMMUNE HEMOLYSIS Warm TypeUsually IgG antibodies.Fix complement only to level of C3,if at all.Immunoglobulin binding occurs at all temps.Fc receptors/C3b recognized by macrophages; Hemolysis primarily extravascular.70% associated with other illnesses.Responsive to steroids/splenectomy.
83Treatment of Autoimmune Hemolytic Anemia September 9, :00 amTreatment of Autoimmune Hemolytic AnemiaTreat underlying disease if indicatedPrednisone (1 mg/kg/day for 2 weeks, then taper)SplenectomyOtherImmunosuppressive agents (Rituximab)IVIGSimilar approach to ITP
89AUTOIMMUNE HEMOLYSIS Cold Type September 9, :00 amAUTOIMMUNE HEMOLYSIS Cold TypeMost commonly IgM mediated.Antibodies bind best at 30º or lower.Fix entire complement cascade.Leads to formation of membrane attack complex, which leads to RBC lysis in vasculature.Typically only complement found on cells.90% associated with other illnesses.Poorly responsive to steroids, splenectomy; but responsive to plasmapheresis&immunosuppressive.
99DRUG-INDUCED HEMOLYSIS Immune Complex Mechanism September 9, :00 amDRUG-INDUCED HEMOLYSIS Immune Complex MechanismDrug & antibody bind in the plasmaImmune complexes eitherActivate complement in the plasma, orSit on red blood cellAntigen-antibody complex recognized by RE systemRed cells lysed as “innocent bystander” of destruction of immune complexREQUIRES DRUG IN SYSTEM.DAT:IgG(-),complement(+).
100DRUG-INDUCED HEMOLYSIS Haptenic Mechanism September 9, :00 amDRUG-INDUCED HEMOLYSIS Haptenic MechanismDrug binds to & reacts with red cell surface proteins.Antibodies recognize altered protein, ± drug, as foreign.Antibodies bind to altered protein & initiate process leading to hemolysis: opsonizes the cells for destruction by splenic macrophages.DAT:IgG(+),complement(-).
101DRUG-INDUCED HEMOLYSIS True Autoantibody Formation September 9, :00 amDRUG-INDUCED HEMOLYSIS True Autoantibody FormationCertain drugs appear to cause antibodies that react with antigens normally found on RBC surface, and do so even in the absence of the drug.DAT:IgG(+),complement(-).
103HEMOLYTIC ANEMIA Summary September 9, :00 amHEMOLYTIC ANEMIA SummaryMyriad causes of increased RBC destruction.Marrow function usually normal.Often requires extra folic acid to maintain hematopoiesis.Anything that turns off the bone marrow can result in acute, life-threatening anemia.