Hemolysis

1. Hemolysis • Increased cell destruction • Rate of destruction exceeds the capacity of the bone marrow to produce red blood cells (RBC) • Normal RBC survival time is 110-120 days • Approximately 1% of RBC are removed each day and replaced by the marrow to maintain the RBC count

2. Hemolysis • During hemolysis RBC survival is shortened and increased marrow activity results in a heightened reticulocyte percentage • Hemolysis can be divided into two • Intravascular hemolysis • Extravascular hemolysis

3. Hemolysis • Extravascular hemolysis • The degradation of Hb results in the biliary excretion of heme pigments and increased fecal urobilinogen • Gallstones composed of calcium bilirubinate may be formed in children as young as 4 years of age

4. Hemolysis • Intravascular hemolysis • Hb binds to haptoglobin and hemopexin both of which are reduced • Oxidized heme binds to albumin to form methemalbumin which is increased • When the capacity of these binding molecules is exceeded, free Hb appears in the plasma (evidence of intravascular hemolysis) • When the tubular reabsorbtive capacity of kidneys for Hb is exceeded free Hb appears in the urine

5. Hemolytic anemia • Hemolysis • Increased cell destruction • A feature of hemolytic anemia is a reduction in the normal red cell survival of 120 days • The premature destruction of RBC may result from corpuscular abnormalities such as • Hb defects, abnormalities of RBC enzymes or defects of RBC membrane • Other defects may result from extracorpuscular abnormalities and may be due to immune or non-immune mechanisms

6. Hemolytic anemia • The approach to the diagnosis of hemolytic anemia should include • Consideration of the clinical features suggesting hemolytic disease • Demonstration of the presence of hemolytic process by laboratory means • Establishment of the presice cause of the hemolytic anemia by special hematologic investigations

7. Hemolytic anemia-Clinical features(1) • The following clinical features suggest hemolysis • Age: anemia and jaundice in an Rh(+) infant born to a Rh(-) mother or a group A or B infant born to a group O mother • History of anemia, jaundice or gallstones in family • Persistent/ recurrent anemia associated with reticulocytosis • Anemia unresponsive to hematinics • Intermittent/persistent indirect hyperbilirubinemia

8. Hemolytic anemia-Clinical features(2) • Splenomegaly • Hemoglobinuria • Presence of multiple gallstones • Chronic leg ulcers • Development of anemia or hemoglobinuria after exposure to certain drugs • Dark urine

9. Hemolytic anemia-laboratory findings • Reduced cell survival and evidence of accelerated Hb catabolism • Evidence of increased erythropoiesis

10. Hemolytic anemia-laboratory findings • Accelerated Hb catabolism • Extravascular • Raised unconjugated bilirubin • Raised fecal and urinary urobilinogen • Intravascular • Hemoglobinuria • Low/absent plasma haptoglobin • Raised plasma methemalbumin

11. Hemolytic anemia-laboratory findings • Increased erythropoiesis (response to a reduction in Hb) • Reticulocytosis • Increased MCV • Increased normoblasts in peripheral blood • Spesific morphological abnormalities • Sickled cells, target cells, spherocytes • Erhytroid hyperplasia of bone marrow • Expansion of marrow space • Prominence of frontal bones, broad cheek bones, widened intratrabecular spaces, hair-on-end appearance of skull radiographs

12. macrocytes normal Target cells Hypochromic, microcytes schistocytes

13. Tests used to establish a spesific cause of hemolytic anemia (1) • Membrane defects (Hereditary spherocytosis, elliptocytosis, stomatosis, acantocytosis) • Blood smear • Increased RBC osmotic fragility • (spherocytes lyse in higher concentrations of saline than normal RBC) • Autohemolysis at 24 and 48 hours • Enzyme defects (G6PD and pyruvate kinase) • Heinz body preparation • Autohemolysis test • Screening tests for enzyme deficiencies

14. Tests used to establish a spesific cause of hemolytic anemia (2) • Hemoglobin defects (sickle cell disease, thalassemias) • Blood smear, sickle cell, target cell • Sickling test • Hemoglobin electrophoresis • HbF determination

15. Tests used to establish a spesific cause of hemolytic anemia (3) • Immune hemolytic anemia • Isoimmune • Mismatched blood transfusion • Hemolytic disease of the newborn • Autoimmune • Action of Ig • Idiopathic, secondary to number of conditions • Coombs’ test (+)

16. Tests used to establish a spesific cause of hemolytic anemia (4) • Non-immune hemolytic anemia • Infections, drugs, underlying hematologic disease- microangiopathic HA, hypersplenism • Coombs’ test (-)

17. Congenital hemolytic anemias ıntracorpuscular • Membrane defects • Hereditary spherocytosis(HS) • Enzyme defects • G6PD deficiency • Hemoglobin defects • - thalassemia (quantitative hemoglobinopathies) • HbS (qualitative hemoglobinopathies) • Hemolytic disease of the newborn (isoimmune)

18. Hereditary spherocytosis • Familial hemolytic disorder • Marked heterogenicity of clinical features • Asymptomatic condition • Fulminant hemolytic anemia • The morphologic hallmark of HS • Microspherocyte • Caused by loss of membrane surface area • Abnormal osmotic fragility

19. Hereditary spherocytosis • HS usually is transmitted as an autosomal dominant trait • An autosomal recessive mode of inheritance also occurs • 20-25% of all HS cases • HS is encountered worldwide

20. Hereditary spherocytosis • An intrinsic genetic defect causes defects in membrane proteins • The major complications • Aplastic or megaloblastic crisis • Hemolytic crisis • Cholecystitis and cholelithiasis • Severe neonatal hemolysis

21. Hereditary spherocytosis- Pathophysiology • HS erythrocytes are caused by membrane protein defects resulting in cytoskeleton instability • Four abnormalities in red cell membrane proteins have been identified • Spectrin deficiency alone (most common) • Combined spectrin and ankyrin deficiency • Band 3 deficiency(10-20% of patients) • Protein 4.2 defects (common in Japan)

22. Hereditary spherocytosis- Pathophysiology • Spectrin deficiency • Loss of erythrocyte surface • Spherical RBC • Culled rapidly from the circulation by the spleen • Splenomegaly • Hemolysis primarily confined to the spleen • Extravascular hemolysis • Biochemical spectrin deficiency and the degree of spectrin deficiency are reported to correlate with the extent of spherocytosis, the degree of abnormality on osmotic fragility test results and the severity of hemolysis

24. Hereditary spherocytosis- Pathophysiology • Ankyrin defects • Ankyrin is the principal binding site for spectrin on RBC membrane • A proportional decrease in spectrin content occurs although spectrin synthesis is normal • 75-80% of patients with autosomal dominant HS have combined spectrin and ankyrin deficiency • Deletion of chromosome 8 are shown to have a decrease in RBC ankyrin content

25. Hereditary spherocytosis- Clinical findings • Anemia • Jaundice • Splenomegaly Clinical features of HS

26. Hereditary spherocytosis- Clinical findings(2) • Anemia or hyperbilirubinemia may be of such magnitude as to require exchange transfusion in the neonatal period • Anemia is mild to moderate • Sometimes severe/not present • In patients with mild HS cholelithiasis may be the first sign of underlying disease • Moderate HS (most common, 60-75%) • Mild HS (20-30%) • Severe HS (5%, requires RBC transfusions)

27. Hereditary spherocytosis- Laboratory findings • Minimal or no anemia • Reticulocytosis • Increased MCHC • Spherocytes on the peripheral blood smear • Howell-Jolly bodies may be seen • Hyperbilirubinemia • Abnormal osmotic fragility test • hemolysis of HS cells may be complete at a solute concentration that causes little or no lysis of normal cells • LDH increased • Increased unconjugated bilirubin • Looking for abnormalities in spectrin, ankyrin, band 3 (not routine)

28. Hereditary spherocytosis- Treatment • Neonates • Phototherapy/exchange transfusion • Aplastic crisis • RBC transfusion • Folic acid supplementation to prevent megaloblastic crisis • Splenectomy (after 6 years of age) • Increased Hb level • Decreased reticulocyte count • Appereance of Howell-Jolly bodies and target cells • Thrombocytosis

29. Glucose 6 phosphate dehydrogenase deficiency (G6PD) • X-linked disorder • Homozygous women are found in populations in which the frequency of G6PD is high • Heterozygous carrier women can develop hemolytic attacks • Polymorphic with more than 300 reported variants • The highest prevalance rates are found in tropical Africa, the Middle East, some areas of Mediterranean (severe forms)

30. Glucose 6 phosphate dehydrogenase deficiency- Pathophysiology • G6PD enzyme catalyzes the oxidation of glucose-6-phosphate to 6-phosphogluconate while reducing the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP+) to nicotinamide adenine dinucleotide phosphate (NADPH)

32. Glucose 6 phosphate dehydrogenase deficiency- Pathophysiology • NADPH • Protects the cells against oxidative stress • Required cofactor in many biosynthetic reactions • Maintains glutathion in its reduced form • Glutathion acts a scavanger for dangerous oxidative metabolites in the cell • Converts harmful hydrogen peroxide to water with the help of glutathion peroxidase

33. Glucose 6 phosphate dehydrogenase deficiency- Clinical findings • The most common clinical feature is no symptoms • Symptomatic patients • Neonatal jaundice • Appears by age 1-4 days • Often requires exchange transfusion • Acute hemolytic anemia • Results from stress factors such as oxidative drugs or chemicals, infection or ingestion of fava beans • Jaundice and splenomegaly may be present during crisis

34. Glucose 6 phosphate dehydrogenase deficiency- Laboratory findings • Anemia • Reticulocytosis • Activity of G6PD is low (after hemolysis) • Indirect hyperbilirubinemia • Serum haptoglobin levels will be decreased • Formation of bodies which consist of denaturated hemoglobin • Heinz body

35. Glucose 6 phosphate dehydrogenase deficiency- Treatment • Avoid oxidant drugs • Antimalarial drugs, nitrofurantoin, nalidixic acid, ciprofloxacin,methylene blue, chloramphenicol, phenazopyridine, vit K analogs, sulfonamides, acetanilid, doxorubicine, isobutyl nitratre, naphtalene, phenylhydrazine, pyridoxin • Exchange transfusion • RBC transfusion

36. Thalassemia (Cooley’s anemia, Mediterranean Anemia) • Genetically determined defect in Hb synthesis • An inability to manufacture sufficient quantities of globin chains • In the adult there are 3 Hb types normally present • Hb A 22 (95% of total) • Hb A2 22 (3% of total) • HbF 22 (2% of total) • During fetal life the majority of Hb • During embryonic life at least 2 different Hbs are produced • Gowers 2 22 chains • Gowers 1 4 chains • The manufacture of each of these chains is controlled by spesific genes

37. Thalassemia • In thalassemia there is a genetic failure in the production of globin chains • Failure of production of  and  chains is the most common •  thalassemia • a failure of beta chain production •  thalassemia • a failure of alpha chain production

38. Beta Thalassemia • The genes controlling beta chain production are located on chromosome 11 •  thalassemia major • If both genes fail •  thalassemia minor • If only one gene fails

39. Beta Thalassemia minor (Heterozygous) (B+) • Most common of thalassemias • Beta chain production is less than normal • Alpha chain production continues at a near normal rate • Decreased level of HbA • Excess alpha chains stimulates the increased production of delta chains • Increased amount of HbA2 • Rate of gamma chain production is greater • Increased amount of HbF

40. Beta Thalassemia minor • These patients are not severely anemic • These patients can be provided appropriate genetic counselling • Hb, Hct are decreased • RBC count is not as low as the Hb and Hct • Bone marrow produce the cells but cannot fill them with Hb • RBCs are microcytic and hypochromic • Normal RDW

41. Beta Thalassemia minor • MCV is slightly decreased • MCH is decreased • MCHC is normal • WBC count is normal • Reticulocyte count is relatively increased • Bone marrow is either normal or undergoes slight erythroid hyperplasia • Serum iron,ferritin is normal • Bilirubin slightly increase due to intramedullary hemolysis

42. Beta Thalassemia minor • Hb studies • HbA decreased • HbA2 increased • HbF slightly increased to normal

43. Beta Thalassemia major(homozygous)(B0) • Complete failure of beta chain production • Raised levels of HbA2 and HbF • HbF has a very high affinity for oxygen (poor oxygen deliverer) • Only functional Hb is HbA2 • The patient is hypoxic • İncreased erythropoietin production • Stimulates the marrow to maximum • Typical facial appereance • Splenomegaly • Extramedullary hemopoiesis

44. Beta Thalassemia major • Patients develop a life threatening anemia by one or two months (mostly often 6 months) • Severe anemia (Hb:2-3 mg/dl) • Hct and RBC count are also decreased • MCV, MCH, MCHC are all decreased • RDW is increased • Hypochromic microcytic RBC • Anisocytosis, poikilocytosis, target cells • Reticulocytosis • WBC is increased at the beginning

45. Beta Thalassemia major • Bone marrow undergoes erythroid hyperplasia • Serum Fe increased/normal • Ferritin increased/normal • Hb electrphoresis • HbA decreased • HbA2 variable • HbF increased

46. Beta Thalassemia major • The patients must be supported with blood transfusions which result in iron overload • Unless iron is removed with appropriate chelation therapy these patients die of hemosiderosis • Splenectomy • When the yearly transfusion requirement of packed red cells exceeds 200-250 ml/kg • Bone marrow transplantation

47. Alpha thalassemia • Four genes coding for alpha chain production • These genes are located on chromosome 6 • There are at least five forms of alpha thalassemia depending on the number and location of abnormal genes

48. Hydrops fetalis- Homozygous alpha thalassemia • All genes are abnormal • There is no alpha chain production • No HbF production and death in utero • At autupsy the cord blood shows severe anemia • There is no HbA and HbF on electrophoresis • most of the Hb is HbBart’s which consists of 4 gamma chains

49. Hemoglobin H disease • Three genes are abnormal and one gene is coding for alpha chains • Limited production of HbF in utero and HbA after birth • The excess gamma chains form HbBart’s and the excess beta chains form HbH • Unstable hemoglobins precipitate in the cell • Premature destruction in the marrow and spleen with splenomegaly • Infant is anemic at birth • RBC and hct count are also decreased

50. Hemoglobin H disease • MCV, MCHC, MCH decreased • RDW is increased • Microcytosis, hypochromia • Reticulocyte count is slightly increased • Bone marrow undergoes erythroid hyperplasia • Serum iron,ferritin increased • Hb electrophoresis • Hb Bart’s increased at birth • Hb Bart’s 2-10% later • HbH 5-40% • HbA and A2 decreased