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HEMOLYTIC ANEMIAS A.Basi A.Basi ADULT HEMATOLOGIST ,ONCOLOGIST ADULT HEMATOLOGIST ,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES IRAN UNIVERSITY OF MEDICAL SCIENCES

HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

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Page 1: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

HEMOLYTIC ANEMIAS

A.BasiA.BasiADULT HEMATOLOGIST ,ONCOLOGISTADULT HEMATOLOGIST ,ONCOLOGIST

IRAN UNIVERSITY OF MEDICAL IRAN UNIVERSITY OF MEDICAL SCIENCES SCIENCES

Page 2: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

Definition

• Hemolysis is defined as a reduction in the average life span of the red cell due to destruction of erythrocytes in the peripheral circulation.

Page 3: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

AnemiaAnemia

Erythrocyte senescence and Erythrocyte senescence and destruction:destruction:

Life span Life span –– 1 2 0 days1 2 0 days

Old, rigid erythrocytes are phagocyzed by theOld, rigid erythrocytes are phagocyzed by the

macrophages in the spleenmacrophages in the spleen

Globin chains are degraded into amino acidsGlobin chains are degraded into amino acids

Heme is degraded into bilirubin and excreted Heme is degraded into bilirubin and excreted

by the liverby the liver

I ron is released and reutilizedI ron is released and reutilized

Page 4: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

Definition

• 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.

Page 5: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

• Anemia, 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.

Definition

Page 6: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

• With optimal marrow compensation, the survival of red cells in the circulation can theoretically decrease from the normal 120 days to as few as 15-20 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.

Definition

Page 7: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

• When red cell survival is so short that anemia develops despite a vigorous

erythropoietic response,the term hemolytic anemia is appropriate.

Definition

Page 8: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

There are two mechanisms of hemolysis:

1.Intravascular hemolysis: is the destructionof 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.

Pathophysiology:

Page 9: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

HEMOLYTIC ANEMIA

• Anemia of increased destruction: Normochromic,normocytic anemia. Shortened RBC survival. Reticulocytosis - Response to increased RBC

destruction. Increased indirect bilirubin. Increased LDH.

Page 10: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES
Page 11: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

Reticulocytes

• Under 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.

Page 12: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

Reticulocytes

• Reticulocytes 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.

Page 13: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

Reticulocytes

• A 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.

Page 14: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

Reticulocyte Count

• Relative reticulocyte count % of all RBC (normal 0.8-1.5%)

• Absolute reticulocyte count Relative reticulocyte count x RBC count Normal 50,000-75,000/µl Examples:

1.1% x 4.96 x106 = 55,000/l

12.2% x 2.05 x106 = 250,000/l

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Page 16: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

The reticulocyte count(kinetic approach)

• Increased reticulocytes (greater than 2-3% or 100,000/mm3 total) are seen in blood loss and hemolytic processes, although up to 25% of hemolytic anemias will present with a normal reticulocyte count due to immune destruction of red cell precursors.

• Retic counts are most helpful if extremely low (<0.1%) or greater than 3% (100,000/mm3 total).

Page 17: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

Corrected 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%):

Page 18: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

RPI = 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.

Page 19: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

The reticulocyte count

• To be useful the reticulocyte count must be adjusted for the patient's hematocrit. Also when the hematocrit is lower reticulocytes are released earlier from the marrow so one can adjust for this phenomenon. Thus:

• Corrected retic. = Patients retic. x (Patients Hct/45) • Reticulocyte index (RPI) = corrected retic.

count/Maturation time (Maturation time = 1 for Hct=45%, 1.5 for 35%, 2 for 25%, and 2.5 for 15%.)

• Absolute reticulocyte count = retic x RBC number.

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Page 21: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

RPI = corrected reticulocyte count / correction factor

• RPI > 3: Good marrow response (hyperproliferative)

• RPI < 2: Inadequate response (hypoproliferative)

• RPI > 2 but < 3 :Appropriate for mild anemia (hemoglobin >10–11 g/dL) but borderline for more severe anemia

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Page 24: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

HEMOLYTIC ANEMIA

• INTRACORPUSCULAR (intrinsic) HEMOLYSIS Membrane Abnormalities Metabolic Abnormalities Hemoglobinopathies

• EXTRACORPUSCULAR HEMOLYSIS Nonimmune Immune

Page 25: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

HEMOLYTIC ANEMIA

Mature 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.

Page 26: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

HEMOLYTIC ANEMIA

• Metabolic 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.

Page 27: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

HEMOLYTIC ANEMIA

• Hemoglobin 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.

Page 28: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

HEMOLYTIC ANEMIAMembrane Defects

• Cytoskeletal defects Hereditary spherocytosis

• Membrane permeability defects Hereditary stomatocytosis

• Increased sensitivity to complement Paroxysmal nocturnal hemoglobinuria

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Page 30: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

The 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.

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Pathophysiology

• Defective 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).

Page 33: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

Hereditary 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.

Page 34: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES
Page 35: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

Clinical 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.

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Page 37: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

Diagnosis 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.

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Page 39: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

Osmotic 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

Page 40: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

HEREDITARY SPHEROCYTOSISOsmotic Fragility

0

20

40

60

80

100

0.3 0.4 0.5 0.6

NaCl (% of normal saline)

% H

emo

lysi

s

Normal HS

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Page 43: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

Hereditary Spherocytosis

• Autosomal dominant disorder

• Abnormality in RBC membrane protein

• Clinical and laboratory findings Splenomegaly Chronic hemolytic anemia Spherocytes on peripheral smear

• Treatment Splenectomy

Page 44: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

Hereditary Elliptocytosis

• Autosomal dominant

• Defective 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.

Page 45: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES
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HEMOLYTIC ANEMIAMembrane abnormalities - Enzymopathies

• Deficiencies in Hexose Monophosphate Shunt Glucose 6-Phosphate Dehydrogenase

Deficiency

• Deficiencies in the EM Pathway Pyruvate Kinase Deficiency

Page 47: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

Glucose 6-Phosphate DehydrogenaseFunctions

• 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.

Page 48: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

Glucose 6-Phosphate Dehydrogenasedeficiency

• 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.

Page 49: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

Glucose-6-phosphate dehydrogenase deficiency

Page 50: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

Glucose 6-Phosphate Dehydrogenasedeficiency

• 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.

Page 51: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

Glucose 6-Phosphate DehydrogenaseDifferent Isozymes

0

0.2

0.4

0.6

0.8

1

0 20 40 60 80 100 120

RBC Age (Days)

G6P

D A

ctiv

ity

(%)

Normal (GdB) Black Variant (GdA-)Mediterranean (Gd Med)

Level needed for protection vs ordinary oxidative stress

Page 52: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

Glucose 6-Phosphate Dehydrogenase variants

• Class I variants: Severe enzyme deficiency (<10% of normal) and chronic hemolysis• Class II variants: Severe enzyme deficiency, but intermittent instead

of chronic hemolysis(G6PD Mediterranean).• Class III variants: Mild to moderate enzyme deficiency (10–60% of normal), with intermittent hemolysis usually precipitated by infection

or oxidative drugs or chemicals(African variant).• Class IV variants: Normal enzyme activity and no anemia or

hemolysis• Class V variants: Increased enzyme activity, without anemia or

hemolysis

Page 53: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

Glucose 6-Phosphate DehydrogenaseFunctions

• Regenerates NADPH, allowing regeneration of glutathione

• Protects against oxidative stress• Lack of G6PD leads to hemolysis during

oxidative stress Infection Medications Fava beans

• Oxidative stress leads to Heinz body formation, extravascular hemolysis.

Page 54: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

Glucose-6-Phosphate Dehydrogenase Deficiency

• Sex-linked disorder• Effects > 200 million people• Hemolytic anemia occurs in the presence

of stress (infection or drugs) African form - mild hemolysis Mediterranean form - more severe

• Unique sensitivity to fava beans

Page 55: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

• the 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.

Page 56: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

Diagnosis 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.

Page 57: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

Diagnosis 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.

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Treatment 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.

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Pyruvate Kinase Deficiency

• The most common enzyme deficiency in the Embden-Meyerhof (glycolytic) pathway .

• However, it is far less common than G6PD deficiency

• Autosomal 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.

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Pathophysiology

• Erythrocytes 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.

Page 63: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

Diagnosis 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.

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Treatment 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%).

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HEMOLYTIC ANEMIACauses

• INTRACORPUSCULAR HEMOLYSIS Membrane Abnormalities Metabolic Abnormalities Hemoglobinopathies

• EXTRACORPUSCULAR HEMOLYSIS Nonimmune Immune

Page 67: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

EXTRACORPUSCULAR HEMOLYSISNonimmune

• Mechanical: 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.

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Microangiopathic Hemolytic AnemiaCauses

• Vascular abnormalities Thrombotic thrombocytopenic purpura Renal lesions

• Malignant hypertension• Glomerulonephritis• Preeclampsia• Transplant rejection

Vasculitis• Polyarteritis nodosa• Rocky mountain spotted fever• Wegener’s granulomatosis

Page 69: HEMOLYTIC ANEMIAS A.Basi ADULT HEMATOLOGIST,ONCOLOGIST IRAN UNIVERSITY OF MEDICAL SCIENCES

Microangiopathic Hemolytic AnemiaCauses - #2

Vascular abnormalities• AV Fistula• Cavernous hemangioma

• Intravascular coagulation predominant Disseminated intravascular coagulation

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HEMOLYTIC ANEMIA - IMMUNE

• Drug-Related Hemolysis

• Alloimmune Hemolysis Hemolytic Transfusion Reaction Hemolytic Disease of the Newborn

• Autoimmune Hemolysis Warm autoimmune hemolysis Cold autoimmune hemolysis

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IMMUNE HEMOLYTIC ANEMIAGeneral Principles

• All require antigen-antibody reactions• Types of reactions dependent on:

Class of Antibody Number & Spacing of antigenic sites on cell Availability of complement Environmental Temperature Functional status of reticuloendothelial system

• Manifestations Intravascular hemolysis Extravascular hemolysis

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IMMUNE HEMOLYTIC ANEMIAGeneral Principles - 2

• Antibodies combine with RBC, & either1. Activate complement cascade, &/or2. Opsonize RBC for immune system

• If 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.

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IMMUNE HEMOLYTIC ANEMIACoombs Test - Direct

• Looks 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.

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IMMUNE HEMOLYTIC ANEMIACoombs Test - Indirect

• Looks 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.

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Anti-Globulin (Coombs) Testing

Direct antiglobulin testing

Indirect antiglobulin testing

Patients RBCs

Patients serum

Anti-C3bAnti-IgG

+

RBCs

+

Anti-IgG

+

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Autoimmune Hemolytic Anemia

• Warm antibodies (IgG-mediated) Primary 45% Secondary 40%

• Lymphoproliferative disease• Connective tissue disease• Infectious disease

Drug-induced 15%

• Laboratory testing Normocytic/macrocytic anemia Peripheral smear: spherocytosis

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AUTOIMMUNE HEMOLYSISWarm Type

• Usually 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.

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Treatment of Autoimmune Hemolytic Anemia

• Treat underlying disease if indicated

• Prednisone (1 mg/kg/day for 2 weeks, then taper)

• Splenectomy

• Other Immunosuppressive agents (Rituximab) IVIG

• Similar approach to ITP

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AUTOIMMUNE HEMOLYSISCold Type

• Most 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.

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Agglutination Reaction

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IMMUNE HEMOLYSISDrug-Related

• Immune Complex Mechanism Quinidine, Quinine,

Isoniazid,rifampin,phenacetin,tetracyclin, carbamazepine.

• “Haptenic” Immune Mechanism Penicillins, Cephalosporins

• True Autoimmune Mechanism Methyldopa, L-DOPA, Procaineamide,

Ibuprofen,interferon-alfa.

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DRUG-INDUCED HEMOLYSISImmune Complex Mechanism

• Drug & antibody bind in the plasma• Immune complexes either

Activate complement in the plasma, or Sit on red blood cell

• Antigen-antibody complex recognized by RE system

• Red cells lysed as “innocent bystander” of destruction of immune complex

• REQUIRES DRUG IN SYSTEM.• DAT:IgG(-),complement(+).

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DRUG-INDUCED HEMOLYSISHaptenic Mechanism

• Drug 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(-).

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DRUG-INDUCED HEMOLYSISTrue Autoantibody Formation

• Certain 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(-).

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HEMOLYTIC ANEMIASummary

• Myriad 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.