Hematology 425 Increased RBC Destruction Intracorpuscular Defects

1. Hematology 425 Increased RBC DestructionIntracorpuscular Defects Russ Morrison November 3, 2006

2. Intracorpuscular Defects – Hereditary Disorders of Cation Permeability and Volume • RBC hydration is determined by intracellular concentration of Na+ and K+ • If the cation content is increased, water enters the cell and forms a hydrocyte, or stomatocyte • If cation content is decreased water leaves the cell and produces a dehydrated RBC or xerocyte

3. Hereditary Stomatocytosis (Hydrocytosis) • A complex mixture of hemolytic anemias in which hemolysis may be mild to severe • Hydrocytosis is rare and usually inherited in an autosomal dominant pattern • It is characterized morphologically by stomatocytes and biochemically by the failure of the Na+ and K+ pumps and increased intracellular water • In most patients a deficiency of a membrane integral protein called stomatin occurs

4. Stomatocytes

5. Hereditary Stomatocytosis (Hydrocytosis) • Clinical and laboratory findings • Mild, moderate or severe hemolysis • 5 to 50% stomatocytes on the PB smear • Macrocytes may be present • Reduced K+ and increased Na+ in the RBCs • Increased RBC osmotic fragility • Patients with anemia may benefit from splenectomy

6. Acquired Stomatocytosis • Seen frequently on PB smears as a drying artifact • Acute alcoholism, some drug therapy and sometimes, marathon runners show stomatocytosis on PB smears • Rhnull patients may exhibit spherocytes and stomatocytes and osmotic fragility may be increased • It is speculated that Rh antigens associated with the RBC membrane, when lost, affects membrane skeletal stability

7. Hereditary Xerocytosis • A rare autosomal dominant hemolytic anemia • RBCs are dehydrated, showing increased MCH and very decreased osmotic fragility • Disease mechanism is not known • RBCs demonstrate stomatocytes, target cells, spiculated RBCs and macrocytes • 2,3-BPG levels is moderately decreased • Splenectomy does not reduce the hemolysis and level of anemia in these patients

8. Acanthocytosis (Spur Cells) • A distinct form of echinocyte (burr cell) in which the cellular projections vary in width, length and surface distribution • Found in sever liver disease, abetalipoproteinemia, infantile pyknocytosis and anorexia nervosa • May be associated with blood groups and hypothyroidism

9. Acanthocytosis (Spur Cells) • Abetalipoproteinemia is a rare autosomal recessive disorder • Associated with the disorder is retinitis pigmentosa, that often results in blindness • Disease usually progresses to death by the time a patient reaches 20s or 30s • 50-100% of RBCs will be acanthocytes • Affected persons have mild hemolytic anemia and normal RBC indices

10. Acanthocytosis (Spur Cells) – Scanning EM

11. Acanthocytosis (Spur Cells) • RBCs have normal membrane protein composition but the membrane lipids are abnormal • Abnormal lipids cause a decrease in the lipid fluidity of the RBC membrane and result in the shape change • Shape change is not present in developing NRBCs or reticulocytes, but becomes evident as the RBCs age

12. Acanthocytosis (Spur Cells) • Other causes of acanthocytosis • Blood group association with McLeod phenotype and In(Lu) gene presence in the Lutheran blood group system • Malnutrition as a result of anorexia nervosa and cystic fibrosis • Vitamin E deficient patients may demonstrate acanthocytes on their blood smear

13. RBC Enzymopathies • HAs may also be the result of RBC enzyme problems • Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency is one of the most common RBC enzyme related causes of HA

14. G6PD Deficiency • The gene for G6PD is located on the X chromosome and shows characteristics of X-linked inheritance • Mutations of the associated genes, when inherited, cause G6PD deficiency either by decreasing its in vivo stability or by affecting its enzymatic functions, or maybe both • Different genetic expressions of G6PD have been divided into classes by the WHO based on clinical symptoms and amount of enzyme activity

15. G6PD Deficiency • WHO Classification of G6PD Deficiency • Class I – severe clinical symptoms of chronic, nonspherocytic HA and less than 20% G6PD activity • Class II – mild clinical expression with intermittent hemolysis and less than 10% activity • Class III – mild clinical expression with intermittent hemolysis associated with infection or drugs and 10% to 60% G6PD activity

16. G6PD Deficiency • Class IV – no clinical expression with 100% activity • Class V – no clinical expression and more than 100% activity • Within the group of syndromes, over 400 different genetic mutations and enzymes may exist which demonstrate different electrophoretic patterns

17. G6PD Deficiency • Normal G6PD has been designated G6PD-B • G6PD variants may be designated with a letter, G6PD-A or by geography, G6PD-Mediterranean • G6PD-A is the most common mutation in blacks while G6PD-Med is the most common in whites • Incidence of G6PD-Med among Kurdish Jews ranges from 3% to 50%

18. G6PD Deficiency • Most individuals are asymptomatic except during oxidative stress resulting from drugs, foods (fava beans) or other causes • Heinz bodies typically appear along with membrane skeletal structural abnormalities • Substances that can trigger hemolysis in patients with G6PD deficiency are listed in box 21-2 on page 277 of the text

19. G6PD Deficiency • Clinical and Laboratory Findings • Most G6PD deficient persons are asymptomatic and never discover their deficiency • Clinical manifestation may only be acute hemolysis during oxidative stress resulting from ingestion of drugs or foods • Anemia during crisis may range from moderate to severe • Gives a normochromic normocytic picture

20. G6PD Deficiency • Clinical and Laboratory Findings • During crisis Heinz bodies (denatured Hgb) develop in the RBCs which may be seen using a supravital stain • Reticulocyte count may be as high as 30% • Haptoglobin level is decreased • Free Hgb may be detected in the plasma • WBC count is usually elevated • Platelet count is variable

21. G6PD Deficiency • Clinical and Laboratory Findings • Unconjugated bilirubin level is elevated • Darkly colored urine tests positive for blood • Intact RBCs do not appear in the urine sediment because the color and blood reaction result from hemoglobinuria and hematuria • G6PD levels may be assayed

22. G6PD Deficiency • G6PD enzyme levels may be screened using UV light to detect fluorescence caused by the production of NADPH by G6PD • NADP does not flouresce while NADPH does

23. G6PD Deficiency • Therapy and Prognosis • The main treatment for G6PD deficiency is avoidance of oxidative stressors. Rarely, anemia may be severe enough to warrant a blood transfusion. Splenectomy generally is not recommended. Folic acid and iron potentially are useful in hemolysis, although G6PD deficiency usually is asymptomatic and the associated hemolysis usually is short-lived. Antioxidants such as vitamin E and selenium have no proven benefit for the treatment of G6PD deficiency.

24. Symptoms and Laboratory Evaluation in Patients with G6PD and Acute Hemolysis

25. Medications to avoid with G6PD Incomplete Listing • Acetanilid • Doxorubicin • Furazolidone • Methylene blue • Nalidixic acid • Niridazole • Nitrofurantoin • Phenazopyridine • Primaquine • Sulfamethoxazone

26. Enzymopathies of the Glycolytic Pathway • As a reminder, the mature RBC lacks a nucleus, mitochondria and other organelles and is unable to synthesize proteins and lipids or to perform oxidative phosphorylation • Energy requirements of the mature RBC are met by generation of ATP through glycolysis • Effective glycolysis is essential for the survival of the RBC

27. Enzymopathies of the Glycolytic Pathway • Enzyme deficiencies or abnormalities that diminish the effectiveness of the glycolytic pathway result in hemolysis or other RBC abnormalities • The most common of these disorders is Pyruvate Kinase (PK) deficiency • Seven other defects of enzymes of the E-M pathway are have been described and are listed in Table 21-4 • Our discussion will be directed toward PK Deficiency

28. Pyruvate Kinase Deficiency • PK deficiency accounts for 90% of the enzyme defects of the glycotic pathway • Inherited as an autosomal recessive trait • HA occurs in homozygotes; heterozygotes are not anemic although their RBCs may show some enzyme alterations • Most common in people of Northern European ancestry, but reported world wide • High prevalence in the Amish of Mifflin County, Pennsylvania (traced to 1 immigrant couple)

29. Pyruvate Kinase Deficiency • Mechanism for hemolysis in PK-deficient RBCs is not known • Alterations in PK-deficient RBCS are thought to result in a rigid cell that is removed from the circulation by the macrophages of the spleen and liver • Anemia may be severe as in neonates, requiring exchange transfusions or fully compensated in apparently healthy adults

30. Pyruvate Kinase Deficiency • No specific therapy is available for PK deficiency except supportive treatment and RBC transfusions, as necessary • Splenectomy does not totally correct the hemolysis, but may raise the Hgb level 1 to 3 g/dL, enough to avoid transfusion

31. Paroxysmal Nocturnal Hemoglobinuria (PNH) • Discovered in the late 1800s when a patient was described who had blood in the urine after sleeping • PNH is a hemolytic anemia, but also a myeloproliferative clonal disorder of the bone marrow • PNH results from a mutation in a hematopoietic stem cell • PNH exhibits an intravascular HA resulting from increased susceptibility of the RBCs to complement

32. PNH • The RBC membrane defect present in PNH is also present in the platelets and granulocytes and maybe the lymphocytes • PNH is the only acquired hemolytic disorder caused by an abnormality of the RBC membrane • PNH results from a clonal somatic mutation of a gene on the X chromosome designated phsphatidylinositol glycan, class A (PIGA) • The mutation occurs at the pluripotential stem cell level

33. PNH • In most PNH patients, this abnormal clone of cells coexists with normal hemopoiesis resulting in a dual population of blood cells • Some PNH patients have over 95% cells with the characteristic abnormality of the absence of the GPI-linked proteins on the cell surfaces • This abnormal cell line appears to originate from a damaged BM

34. PNH • Many PNH patients have a prior history of aplastic anemia or pancytopenia that has been induced by drugs or for which the cause is unknown (idiopathic) • The main defect in PNH is an acquired membrane abnormality that causes increased susceptibility of blood cells to complement

35. PNH • The complement connection was made when two complement regulatory proteins, CD55 (decay accelerating factor) and CD59 (membrane inhibitor of reactive lysis) were found to be missing from blood cells in PNH • Because of the lack of these proteins, randomly deposited complement factors and C3 convertase complexes are not removed from the RBC membrane, leading to the formation of the membrane attack complex, pore formation, and RBC lysis

36. PNH • CD55, CD59 and other proteins use a GPI anchor for attachment to the RBC membrane • The entire GPI-linked polypeptide is extracellular • All of the GPI-linked surface proteins that occur normally on blood cells are absent from PNH cells because of the incomplete bioassembly of the GPI anchors

37. PNH • A mutation in the gene designated as PIGA has been identified as the gene defect in PNH • The PIGA gene resides on the X chromosome • PNH RBCs are classified into subpopulations according to their susceptibility to complement-mediated lysis

38. PNH • Flow cytometry is used today to analyze surface expression of the GPI-linked proteins (CD16, CD48, CD55, CD59) • RBCs that are totally deficient in GPI-linked surface proteins are the PNH type III cells • Type I cells have normal amounts of the surface proteins and are normal in their response to complement

39. PNH • The percentage of PNH-III cells determines the intensity of the clinical symptoms • Patients with less than 20% PNH-III cells have mild hemolysis • 20-50% PNH-III cells will show episodic hemolysis (sleep induced) • Patients with more than 50% PNY-III cells will exhibit perpetual hemoglobinemia and hemoglobinuria

40. PNH – Clinical Findings • Up to 30% of PNH patients may have sever aplastic anemia for several years before diagnosis • PNH has been associated with middle age or young adulthood, but can occur at any age • PNH affects both sexes equally • Passage of reddish urine on arising from sleep (hence the name) only occurs in 25% of patients

41. PNH • Some nocturnal hemoglobinuria occurs in most patients • Hemolytic episodes may be triggered by • Lower blood pH during sleep (facilitates complement binding on PNH cells) • Infections • Vaccinations • Transfusions • X-ray contrast dye exposure • Strenuous exercise

42. PNH • Other findings • Hemoglobinuria • Hemosiderinuria • Iron deficiency anemia due to heavy loss of iron in the urine • Infections are common due to impaired neutrophil function • Predisposition to intravascular thrombosis (brain - headaches, portal vein – abdominal pain)

43. PNH • PNH can evolve into acute nonlymph-ocytic leukemia at an incidence of 1-3%, far greater than seen in the general population • The leukemic transformation typically occurs within the first 5 years of the disease

44. PNH – Routine Hematologic Findings • Pancytopenia with a mild to sever anemia, leukopenia and thrombocytopenia • Hgb ranges from below 6 g/dL to normal • Reticulocyte levels are mildly to moderately elevated • Anisocytosis and poikilocytosis is absent • Osmotic fragility is normal • DAT is negative • Moderate neutropenia is present leading to infections which cause 5%-10% of the fatalities in PNH patients • Platelet count is decreased

45. PNH – Routine Hematologic Findings • BM shows normal cellularity with RBC hyperplasia • Iron may be absent from the BM when anemia is severe • Hemosiderinuria is a constant feature of PNH and is diagnostically important • Renal problems may develop in PNH patients due to deposition of iron in the kidneys

46. PNH – Special Tests • Suspect PNH in any patient with idiopathic pancytopenia and acquired nonspherocytic anemia accompanied by reticulocytosis • PNH screening tests include urine hemosiderin determination and the sucrose hemolysis test • If the sucrose hemolysis test is positive, the Ham test (acidified serum lysis test) was used to confirm the diagnosis

47. PNH – Special Tests • PB RBCs and granulocytes are now analyzed by flow cytometry for surface expression of the GPI-linked proteins (CD16, CD48, CD55, CD59) • Figure 21-13 in the txt shows immunophenotyping of PNH cells using flow cytometry

48. PNH – Therapy & Prognosis • Treatment of PNH is supportive with transfusions, antibiotics and anticoagulants • Iron therapy and folate may help alleviate further development of anemias secondary to the PNH • Steroids may decrease hemolysis • Androgenic steroids may be effective in cases with prominent marrow hypoplasia

49. PNH – Therapy & Prognosis • Anticoagulants lessen thrombotic complications • In severe cases, where suitable donors are available, bone marrow transplantation may be curative • Clinical course of PNH varies widely • Occasionally a patient dies with the first few months of diagnosis • Most patients experience a chronic course with severity changing based on the % normal cells and PNH cells present

50. PNH – Therapy & Prognosis • PNH is a very serious disease and most patients die from its complications (thrombotic episodes, infections) • Acute leukemia may be the terminal event experienced by the PNH patient