Hemolysis: premature destruction of the red cells

1. Hemolysis: premature destruction of the red cells RBC normally survive 90 to 120 days in the circulation.

2. Kind of hemolysis: • Extravascular Hemolysis: • RBC prematurely removed from the circulation by macrophages, particularly those of the spleen and liver • intravascular hemolysis: • less commonly, by disruption of their membranes during their circulation ------------------------------------------------- The level of unconjugated bilirubin never exceeds 70 to 85 umol/L (4 to 5 mg/dL), unless liver function is impaired. DR MOKARIAN

3. DR MOKARIAN Hemoglubin Hemeglobulin Protopurphyrin Fe Aminoacide Unconjugated bil Conjugated bil

4. (LDH), particularly LDH-2, is elevated • Serum AST (SGOT) may be somewhat elevated, whereas ALT (SGPT) is not. • Haptoglobin : • High concentration (~1.0 g/L) in the plasma (and serum) • It binds specifically and tightly to the globin in hemoglobin • Cleared within minutes by the mononuclear phagocyte system. • Is decreased in patients with hepatocellular disease and increased in inflammatory states DR MOKARIAN

5. DR MOKARIAN plasma hemoglobin is increased in proportion to the degree of hemolysis Plasma hemoglobin may be falsely elevated due to lysis of RBC in vitro Hemosiderin appears 3 to 4 days after the onset of hemoglobinuria and may persist for weeks after its cessation. glomerular permeability than myoglobin and is less rapidly cleared by the kidneys.

6. the urine is positive with the benzidine reaction: Hemoglobinuria , Hematuria , myoglobinurea The distinction between hemoglobinuria and myoglobinuria: Can best be made by specific tests that exploit immunologic differences Differences in solubility: After centrifugation of an anticoagulated blood specimen, a reddish-brown color (Hb) normal in color (myoglobin) Because of its higher molecular weight, hemoglobin has lower glomerular permeability than myoglobin and is less rapidly cleared by the kidneys. DR MOKARIAN

7. Classification of Hemolytic Anemias DR MOKARIAN Intra corpuscular 1. Abnormalities of RBC interior a. Enzyme defects b. Hemoglobinopathies (Chap. 106) 2. RBC membrane abnormalities a. Hereditary spherocytosis etc. b. Paroxysmal nocturnal hemoglobinuria Heredtary Extra corpuscular c. Spur cell anemia 3. Extrinsic factors a. Hypersplenism b. Antibody: immune hemolysis c. Microangiopathic hemolysis d. Infections, toxins, etc. Acquired In intracorpuscular types of hemolysis, the patient's RBC have an abnormally short life span in a normal recipient (with a compatible blood type), while compatible normal RBC survive normally in the patient. The opposite is true in extracorpuscular types of hemolysis.

8. INHERITED HEMOLYTIC ANEMIAS Defects are often known at the genomic level 1- The membrane 2-The enzyme 3-Hemoglobin Red Cell Membrane Disorders These are usually readily detected by morphologic abnormalities of the RBC on the blood film Three types of inherited RBC membrane abnormalities: 1- Hereditary spherocytosis 2- Hereditary elliptocytosis 3- pyropoikilocytosis 4- hereditary stomatocytosis. . DR MOKARIAN

9. DR MOKARIAN Hereditary spherocytosis • This condition is characterized by : • DEFECT OF ONE PROTEIN OF CYTOSKELATON • LOSS OF RBC MEMBRANE  • S/V DECREASE  • SPHEROCYTOSIS • usually has an autosomal dominant • 1:1000 to 1:4500 • ~20% autosomal recessive inheritance or a spontaneous mutation • The disorder is sometimes clinically apparent in early infancy but often escapes detection until adult life.

10. DR MOKARIAN Hereditary spherocytosis • CLINICAL MANIFESTATIONS • Anemia • anemia is usually mild or moderate and may even be absent in an otherwise healthy individual • erythroid hyperplasia of the bone marrowextramedullary erythropoiesis paravertebral masses visible on chest x-ray • episodes of relative erythroid hypoplasia precipitated by infections, particularly parvovirus, trauma, surgery, and pregnancy • Splenomegaly • Splenomegaly is very common • The hemolytic rate may increase transiently during systemic infections, which induce further splenic enlargement. • Jaundice("congenital hemolytic jaundice“) • Jaundice may be intermittent and tends to be less pronounced in early childhood • pigmented gallstones are common • Chronic leg ulcers

11. Hereditary spherocytosis DR MOKARIAN • Laboratory: • MCV: is usually normal or slightly decreased • MCHC : is increased to 350 to 400 g/L • Osmotic fragility test: increased • PBS :spherocytes are usually detected as small cells without central pallor • They will ordinarily not influence the osmotic fragility test unless they constitute more than 1 or 2% of the total cell population • The autohemolysis test is also useful.

12. Hereditary spherocytosis DR MOKARIAN OSMOTIC FRAGILITY TEST

13. DR MOKARIAN Hereditary spherocytosis

14. Hereditary spherocytosis DR MOKARIAN • PATHOGENESIS : PROTEIN DEFICIENCY • Nearly all patients have a significant deficiency of spectrin • 50% of patients have a defect in ankyrin • 25% of patients have a mutation of protein 3 • remaining 25% have mutations of spectrin, leading to impaired synthesis or self-association • b-spectrin deficiency is generally mild, with dominant inheritance • a-spectrin deficiency is severe, with a recessive inheritance pattern. • "conditioning“: the lipid bilayer is not well anchored when these proteins are defective part of it is lost by vesiculation resulting in a more spherical less deformable cell. • DESTRUCTION ON SPLEEN • "conditioning" produces a subpopulation of hyperspheroidal RBC in the peripheral blood.

15. DR MOKARIAN • DIFERENTIAL DIAGNOSIS: • Immune spherocytosis • Cirrhosis • In clostridial infections, • Certain snake envenomations • Glucose -6-phosphate dehydrogenase (G6PD) deficiency.

16. DR MOKARIAN Hereditary spherocytosis • TREATMENT: • 1- Splenectomy • corrects the anemia, although the RBC defect and its consequent morphology persist • The operative risk is low • splenectomy should be performed in symptomatic individuals • cholecystectomy should not be performed without splenectomy, as intrahepatic gallstones may result • Splenectomy in children should be postponed until age 4 • Polyvalent pneumococcal vaccine should be administered at least 2 weeks before splenectomy • 2- folic acid(1 mg/d) should be administered prophylactically.^

17. Hereditary elliptocytosis : Oval or elliptic RBC are normally found in birds, reptiles, camels, and llamas autosomal dominant trait 1 per 4000 to 5000 people (similar to that of hereditary acquired elliptocytosis : rarely in patients with MDS PATOGENESIS : 1- Spectrin Def. 2- protein 4.1 Def. PRESENTATION :MILD ANEMIA (HB>120 RET<4% In 10 to 15% of patients with more severe abnormalities, the rate of hemolysis is substantially increased, with median survival times of RBC as short as 5 days and reticulocytes ranging up to 20%. RBC destruction occurs predominantly in the spleen, which is enlarged in patients with overt hemolysis Hemolysis is corrected by splenectomy. DR MOKARIAN

18. DR MOKARIAN Hereditary elliptocytosis at least 25% and, more commonly, >75% of RBC are elliptic, with an axial ratio (width/length) of <0.78. Patients with hemolysis frequently have microovalocytes, bizarre-shaped RBC, and RBC fragments, all of which increase in number after splenectomy. The degree of hemolysis does not correlate with the percentage of elliptocytes. Osmotic fragility is usually normal but may be increased in patients with overt hemolysis.

19. DR MOKARIAN • Hereditary pyropoikilocytosis: • rare disorder • bizarre-shaped, microcytic RBC that undergo disruption at temperatures of 44 to 45°C • normal RBC are stable up to 49°C • Hemolysis is usually severe • recognized in childhood, and is partially responsive to splenectomy.

20. Hereditary Stomatocytosis: cup-shaped RBC (concave on one face and convex on the other). This formation results in a slitlike central zone of pallor on dried smears. autosomal dominant pattern. RBC have an increased permeability to sodium and potassium overhydrated stomatocytes, "hydrocytosis“ In some patients, the RBC are swollen with an excess of ions and water and a decreased mean corpuscular hemoglobin concentration (many of these patients lack the RBC membrane protein 7.2 (stomatin) These are true stomatocytes on dried smears. dehydrated stomatocytes, "desiccytosis" or "xerocytosis" In other patients, the RBC are shrunken, with a decreased ion and water content and an increased mean corpuscular hemoglobin concentration Theas assume the morphology of target cells on dried smears Osmotic fragility is increased in overhydrated stomatocytes and decreased in underhydrated stomatocytes RBC lacking Rh proteins (Rhnull cells) are stomatocytic and have a shortened life span Sign & symptom :splenomegaly and mild anemia Treatment: Splenectomy decreases but does not totally correct the hemolytic process.

21. Red Cell Enzyme Defects • The RBC loses its nucleus, ribosomes, and mitochondria and thus its capability for protein synthesis and oxidative phosphorylation. • ATP must be generated from the Embden-Meyerhof pathway to: • Drive the cation pump that maintains the ionic milieu in the RBC. Preservation of hemoglobin iron in the ferrous (Fe2+) state • For the renewal of the lipids in the RBC membrane. • About 10% of the glucose consumed by the RBC is metabolized via the hexose-monophosphate shunt which protects both hemoglobin and the membrane from exogenous oxidants, including certain drugs

22. Red Cell Enzyme Defects Defects in the Embden-Meyerhof Pathway may be restricted to RBC or not Mutations can result in no protein product, a dysfunctional product, or an unstable product These enzymopathies have similar pathophysiologic and clinical features Patients present with a congenital nonspherocytic hemolytic anemia of variable severity The RBC are often relatively deficient in ATP, resulting in a leak of potassium ion out of these cells These RBC are rigid and thus more readily sequestered by the mononuclear phagocyte system. pyruvate kinase (PK) deficiency and hexokinase deficiency are localized to the RBC Glucose phosphate isomerase deficiency and phosphoglycerate kinase deficiency also involve leukocytes

23. Red Cell Enzyme Defects Defects in the Embden-Meyerhof Pathway Individuals with deficiency of triose phosphate isomerase have decreased levels of enzyme in leukocytes, muscle cells, and cerebrospinal fluid, and they have a progressive neurologic disorder. Some patients with phosphofructokinase deficiency have a myopathy. Incidence: defects in the glycolytic pathway PK deficiency : 95% glucose phosphate isomerase deficiency: 4% Other 1%

24. Red Cell Enzyme Defects Defects in the Embden-Meyerhof Pathway A number of different mutations result in PK deficiency. There is considerable variability in the clinical manifestations and laboratory findings among individuals reported as having PK deficiency Most of these patients are compound heterozygotes who have inherited a different defective enzyme from each parent. autosomal recessive pattern: most ofen (the parents are entirely asymptomatic) Phosphoglycerate kinase deficiency is inherited as a sex-linked disorder|(male sever anemia female mild hemolytic anemia )

25. HEMOLYTIC ANEMIAS DR MOKARIAN Red Cell Enzyme Defects • Defects in the Embden-Meyerhof Pathway • CLINICAL MANIFESTATIONS • Anemia jaundice splenomegaly • LABORATORY FINDINGS • PBS normocytic (or slightly macrocytic), normochromic anemia with reticulocytosis. • In those with PK deficiency, bizarre erythrocytes, including spiculated cells, are noted on the peripheral smear, especially after splenectomy. • Spherocytes are usually absent; hence the term congenital nonspherocytic hemolytic anemia • the osmotic fragility of freshly drawn blood is usually normal • AUTOHEMOLYSIS TEST • Incubation brings out an osmotically fragile population of RBC, an abnormality not corrected by the addition of glucose. • specific enzymatic assays

26. HEMOLYTIC ANEMIAS DR MOKARIAN • Red Cell Enzyme Defects • Defects in the Embden-Meyerhof Pathway • TREATMENT: • Most patients do not require therapy • Those with severe hemolysis should be given folic acid (1 mg/d) • Blood transfusions may be necessary during a hypoplastic crisis • Women with PK deficiency may become very anemic during pregnancy • sometimes leading to the diagnosis for the first time • splenectomy • PK • glucose phosphate isomerase • not been proven effective in individuals with other glycolytic enzymopathies.

27. Red Cell Enzyme Defects: Defects in the hexose-monophosphate shunt Glucose metabolism via the hexose-monophosphate shunt Reduced glutathione protecting the sulfhydryl groups of hemoglobin and the RBC membrane from oxidation inherited defect in the hexose-monophosphate shun unable to maintain an adequate level of reduced glutathione in their RBC Hemoglobin sulfhydryl groups become oxidized The hemoglobin precipitates within the RBC forming Heinz bodies.

28. Red Cell Enzyme Defects Defects in the hexose-monophosphate shunt :G6PD DEFICIENCY most common congenital shunt defect defective home for the merozoite over 400 variants of G6PD In most cases, the alteration is a base substitution, leading to an amino acid replacement DIFERENT SEVERITY: nonspherocytic hemolytic anemia without demonstrable oxidant stress (particularly shortly after birth) through hemolytic anemia only when stimulated by marked to mild oxidant stress, to no clinically detectable abnormality TYPE B: The normal G6PD TYPE A+: About 20% of individuals of African descent have a G6PD (that differs by a single amino acid and is electrophoretically distinguishable but functionally normal. . type A- : same electrophoretic mobility as the A+ type, but it is unstable and has abnormal kinetic properties 11% of African American males Mediterranean type more severe than the A- Chinesetype : less severe variant

29. Red Cell Enzyme Defects • Defects in the hexose-monophosphate shunt :G6PD FICIENCY: • sex-linked trait( x linked ) • males (hemizygotes) • female carriers (heterozygotes):Lyon hypothesis • Most female carriers are asymptomatic • G6PD activity normally declines ~50% during the 120-day life span of the RBC • A-Varien( mild non clinical anemia ) • Stess factor:some type of environmental stress • viral and bacterial infections(most often)mechanism is unknown • most commonly sulfa drugs, antimalarials, and nitrofurantoin • Although aspirin is frequently mentioned as a likely offender, it has no deleterious effect in A- individuals. • Metabolic acidosis , toxic

30. Red Cell Enzyme Defects • G6PD FICIENCY : • CLINICAL AND LABORATORY FEATURES • acute hemolytic crisis within hours of exposure to the oxidant stress • leading to hemoglobinuria and peripheral vascular collapse • hemolytic crisis is usually self-limited • Since only the older population of RBC is rapidly destroyed • A- variant, the RBC mass decreases by a maximum of 25 to 30% • laboratory • HCT • rise in plasma hemoglobin • unconjugated bilirubin • decrease in plasma haptoglobin • Heinz bodies, visualized by means of a supravital stain such as crystal violet. • Heinz bodies are usually not seen after the first day or so, since these inclusions are readily removed by the spleen • "bite cells" (RBC that have lost a peripheral portion of the cell) • Multiple bites cause the formation of fragments

31. Red Cell Enzyme Defects Defects in the hexose-monophosphate shunt :G6PD FICIENCY: Mediterranean type G6PD: much lower overall enzyme activity more severe clinical manifestations sensitive to fava beans:favism  he oxidants in Vicia fava are two b-glycosides whose aglycones, when autooxidized, produce oxygen free radicals. Favism is not encountered in individuals with the A- variant.

32. Red Cell Enzyme Defects • Defects in the hexose-monophosphate shunt : • G6PD FICIENCY • The diagnosis of G6PD deficiency: • In any individual, particularly a male of African or Mediterranean descent, who experiences an acute hemolytic episode. • The enzyme activity or the effects of its deficiency. • A false-negative result during a hemolytic episode when the old RBC containing the defective enzyme have already lysed.

33. Red Cell Enzyme Defects • Defects in the hexose-monophosphate shunt :G6PD FICIENCY: • TREATMENT: • no specific treatment is necessary. • Since hemolysis in patients deficient in A- G6PD is usually self-limited • Splenectomy does not benefit • Blood transfusions are rarely indicated • Adequate urine flow • Prevention of hemolytic episodes is best • Infections ought to be treated promptly. • warned about risks posed by oxidant drugs and fava beans • Any patient of African or Mediterranean ancestry about to be given an oxidant drug should be screened for G6PD deficiency

34. Table 108-4. Drugs Causing Hemolysis in Subjects Deficient in G6PD Red Cell Enzyme Defects Antimalarials: Primaquine, pamaquine, dapsone Sulfonamides: Sulfamethoxazole Nitrofurantoin Analgesics: Acetanilid Miscellaneous: Vitamin K (water-soluble form), doxorubicin, methylene blue, nalidixic acid, furazolidone, niridazole, phenazopyridine

35. ACQUIRED HEMOLYTIC ANEMIAS: RBC are made normally but are prematurely destroyed because of damage acquired in the circulation. he exceptions are rare disorders characterized by acquired dysplasia of the cells of the bone marrow

36. Causes of Acquired Hemolytic Anemia II. Immune A. Warm-reactive (IgG) antibody B. Cold-reactive IgM antibody (cold agglutinin disease) C. Cold-reactive IgG antibody (paroxysmal cold hemoglobinuria) D. Drug-dependent antibody 1. Autoimmune 2. Haptene III. Traumatic hemolytic anemia A. Impact hemolysis B. Macrovascular defects¾prostheses C. Microvascular causes 1. Thrombotic thrombocytopenic purpura/hemolytic-uremic syndrome 2. Other causes of microvascular abnormalities 3. Disseminated intravascular hemolysis IV. Hemolytic anemia due to toxic effects on the membrane A. Spur cell anemia B. External toxins 1. Animal or spider bites 2. Metals (e.g., copper) 3. Organic compounds V. Paroxysmal nocturnal hemoglobinuria

37. ACQUIRED HEMOLYTIC ANEMIAS Hypersplenism blood circulatingspleenarterioleswhite pulp to sinuses(98%) red pulpvenous system 1 to 2% "marginal zone" of the lymphatic white pulp cells that occupy this zone are not phagocytic serve as a mechanical filter

38. ACQUIRED HEMOLYTIC ANEMIAS Hypersplenism: The normal spleen retains reticulocytes for 1 to 2 days In infiltrative diseases of the spleen, substantial splenomegaly may exist with no apparent hemolysis; inflammatory and congestive splenomegaly is commonly associated with modest shortening of RBC survival time, along with more marked granulocytopenia and thrombocytopenia.

39. ACQUIRED HEMOLYTIC ANEMIAS:Immunologic Causes of Hemolysis The Coombs antiglobulin test is the major tool for diagnosing autoimmune hemolysis. neither IgG nor complement may be found on the RBC of the patient (Coombs-negative immune hemolytic anemia) IgG anti c3 = warm Ab IgM = cold Ab

40. Table 108-6. Use of the Direct Coombs Test in Diagnosing the Cause of Autoimmune Hemolytic Anemia Reaction with Anti-IgG Anti-C3 Causes Yes No Antibodies to Rh proteins, hemolysis caused by a-methyldopa or penicillin Yes Yes Antibodies to glycoprotein antigens, SLE No Yes Cold-reacting antibodies (agglutinins or Donath-Landsteiner antibody), most drug-related antibodies, IgM antibodies, IgG antibodies of low affinity, activation of complement by immune complexes ACQUIRED HEMOLYTIC ANEMIAS

41. ACQUIRED HEMOLYTIC ANEMIAS: Immunologic Causes of Hemolysis "Warm" antibodies nearly always IgG occasionally, they are IgA and rarely IgM Hemolysis due to autologous antibodies is called autoimmune hemolytic (or immunohemolytic) anemia, warm antibody type

42. ACQUIRED HEMOLYTIC ANEMIAS:Immunologic Causes of Hemolysis Warm" antibodies CLINICAL MANIFESTATIONS occurs at all ages, but it is more common in adults, particularly women. one-fourth an underlying disease: next slide The presentation quite variable severe anemia [hemoglobin levels 60 to 100 g/L and reticulocyte counts 10 to 30% spherocytosis Splenomegaly fulminant hemolysis associated with hemoglobinemia, hemoglobinuria, and shock; this syndrome may be rapidly fatal unless aggressively treated. The direct Coombs test is positive in 98% of patients usually IgG is detected with or without C3 Evans's syndrome : immune throbocytopenia + anemia Occasionally, venous thrombosis occurs

43. ACQUIRED HEMOLYTIC ANEMIAS:Immunologic Causes of Hemolysis CLINICAL MANIFESTATIONS Hemolysis due to Antibodies WARM-ANTIBODY IMMUNOHEMOLYTIC ANEMIA 1. Idiopathic , Lymphomas: Chronic lymphocytic leukemia, non-Hodgkin's lymphomas, Hodgkin's disease (infrequent) ,SLE and other collagen-vascular diseasesDrugs , a-Methyldopa type (autoantibody to Rh antigens) , Penicillin type (stable hapten)Quinidine type (unstable hapten) , Postviral infectionsOther tumors (rare) COLD-ANTIBODY IMMUNOHEMOLYTIC ANEMIA cCold agglutinin disease , Acute: Mycoplasma infection, infectious mononucleosis , Chronic: Idiopathic, lymphoma , Paroxysmal cold hemoglobinuria

44. ACQUIRED HEMOLYTIC ANEMIAS:Immunologic Causes of Hemolysis Warm" antibodies PATHOGENESIS IgG antibodies lyse RBC by two mechanisms (1) immune adherence of RBC to phagocytes mediated by the antibody and by complement components that become fixed to the membrane (by far the more important mechanism of destruction), and (2) complement activation. formation of spherocytes If internalization is only partial, the RBC membrane is removed, resulting in the formation of spherocytes, which are destroyed in the spleen. Complement-mediated immune adherence involves the interaction of C3b and C4b with receptors on the macrophage;

45. ACQUIRED HEMOLYTIC ANEMIAS ACQUIRED HEMOLYTIC ANEMIAS:Immunologic Causes of Hemolysis Warm" antibodies TREATMENT mild degree of hemolysis usually do not require therapy clinically significant hemolysis:prednisone, 1.0 mg/kg per day) A rise in hemoglobin is frequently noted within 3 or 4 days and occurs in most patients within 1 to 2 weeks Prednisone is continued until the hemoglobin level has risen to normal values, and thereafter it is tapered rapidly to about 20 mg/d, then slowly over the course of several months For chronic therapy with prednisone, alternate-day administration is preferred

46. ACQUIRED HEMOLYTIC ANEMIAS ACQUIRED HEMOLYTIC ANEMIAS:Immunologic Causes of Hemolysis Warm" antibodies TREATMENT More than 75% initial significant in half these patients the disease recurs, either during glucocorticoid tapering or after its cessation Glucocorticoids have two modes of action: an immediate effect due to inhibition of the clearance of IgG-coated RBC by the mononuclear phagocyte system and a later effect due to inhibition of antibody synthesis.

47. ACQUIRED HEMOLYTIC ANEMIAS:Immunologic Causes of Hemolysis Warm" antibodies TREATMENT immunosuppressive drugs A success rate of ~50% Intravenous gamma globulin may cause rapid cessation of hemolysis it is not nearly as effective in this disorder as in immune thrombocytopenia. blood transfusions( problem = selection approprat doner) antibody in this disease is usually a "panagglutinin," reacting with nearly all normal donor cells, cross-matching is impossible adsorb the panagglutinin present in the patient's serum with the patient's own RBC from which antibody has been previously eluted Serum cleared of autoantibody can then be tested for the presence of alloantibody to donor blood groups

48. ACQUIRED HEMOLYTIC ANEMIAS: warm antibody TREATMENT:

49. ACQUIRED HEMOLYTIC ANEMIAS:Immunologic Causes of Hemolysis • Warm" antibodies • PROGNOSIS: • In most patients, hemolysis is controlled • Fatalities occur among three rare subsets of patients: • those with overwhelming hemolysis who die from anemia; • (2) those whose host defenses are impaired by glucocorticoids, splenectomy, and/or immunosuppressive agents; and • (3) those with major thrombotic events coincident with active hemolysis.

50. ACQUIRED HEMOLYTIC ANEMIAS:Immunologic Causes of Hemolysis • Warm" antibodies • Immunohemolytic anemia secondary to drugs • two mechanisms of action: • they induce a disorder identical in almost every respect to warm-antibody immunohemolytic anemia (e.g., a-methyldopa ) • (2) they become associated as haptenes with the RBC surface and induce the formation of an antibody directed against the RBC-drug complex (e.g., penicillin, quinidine).