Hemolytic Anemia: Enzyme Deficiencies

1. 18 Hemolytic Anemia: Enzyme Deficiencies

2. Learning Objectives—Level I At the end of this unit of study, the student should be able to: • Identify the two main pathways by which erythrocytes catabolize glucose. • Explain the role of erythrocyte enzymes in maintaining the cell's integrity, and describe how deficiencies in these enzymes lead to anemia. • Identify the most common erythrocyte enzyme deficiency. continued on next slide

3. Learning Objectives—Level I At the end of this unit of study, the student should be able to: • Describe the inheritance pattern for glucose-6-phosphate dehydrogenase (G6PD). • Explain how the diagnosis of G6PD deficiency is made. • List the tests used to detect G6PD deficiency and describe their principles. continued on next slide

4. Learning Objectives—Level I At the end of this unit of study, the student should be able to: • Recognize the erythrocyte morphology in a Romanowsky-stained blood smear associated with G6PD deficiency. • Identify common compounds that induce anemia in G6PD deficiency.

5. Learning Objectives—Level II At the end of this unit of study, the student should be able to: • Recommend appropriate laboratory testing and interpret results for suspected G6PD deficiency following a hemolytic episode. • Explain the function of glutathione in maintaining cellular integrity. continued on next slide

6. Learning Objectives—Level II At the end of this unit of study, the student should be able to: • Associate the mechanisms of hemolysis with defects in the glycolytic and hexose monophosphate shunt pathways. • Correlate clinical and laboratory findings with the common G6PD isoenzyme variants. continued on next slide

7. Learning Objectives—Level II At the end of this unit of study, the student should be able to: • Diagram the reaction catalyzed by pyruvate kinase, and explain how a defect of this enzyme can cause hemolysis. • Recognize erythrocyte morphology associated with pyruvate kinase deficiency. continued on next slide

8. Learning Objectives—Level II At the end of this unit of study, the student should be able to: • Review and interpret laboratory findings in a case study presentation of G6PD deficiency.

9. Introduction • Mature RBCs • Depend on anaerobic glucose metabolism for energy • RBC enzyme activity stable • Amount of enzyme present for normal cell function is limited

10. Introduction • Mature reticulocytes • Lose nucleus, mitochondria, ribosomes • Can't synthesize protein or undergo oxidative phosphorylation for ATP production • Inherited RBC enzyme deficiency • Compromise integrity of cell membrane or Hb • Hemolysis

11. Introduction • Two most common enzyme defects: • Glucose-6-phosphate dehydrogenase (G6PD) • Affects hexose monophosphate shunt • Found more frequently • Pyruvate kinase (PK) • Affects glycolytic pathway (Embden-Meyerhof) • Second most common

12. Table 18-1 Erythrocyte Enzyme Deficiencies Associated with Congenital (Chronic) Nonspherocytic Hemolytic Anemia

13. Hexose Monophosphate Shunt • HM shunt catabolizes 10% of the glucose • Maintains adequate levels of reduced glutathione (GSH) • GSH maintained by the conversion of NADPH→NADP • NADP is reduced back to NADPH by G6PD continued on next slide

14. Hexose Monophosphate Shunt • HM shunt catabolizes 10% of the glucose • GSH • Protects RBC from oxidant damage • Maintains HB in the reduced functional state • Preserves vital cellular enzymes

15. Figure 18-1 G6PD is needed for maintaining adequate quantities of glutathione (GSH), an important buffer to oxidants within the erythrocyte. As GSH reduces H2O2 to H2O, it is oxidized (GSSG). G6PD generates NADPH in the conversion of glucose-6-phosphate to 6-phosphogluconate. NADPH, in turn, regenerates reduced glutathione from oxidized glutathione.

16. Hexose Monophosphate Shunt • Enzyme deficiencies in HMP shunt • Oxidation of hemoglobin • Formation of Heinz bodies • Spleen removes Heinz bodies • Extravascular hemolysis • Most common enzyme deficiency • G6PD deficiency

17. Glycolytic Pathway • RBC energy—glycolysis • 90% of the glucose is utilized by this pathway • Maintains adequate levels of ATP, needed for • Active cation transport across the cell membrane • Maintains membrane deformability • Maintains RBCs' bioconcave shape continued on next slide

18. Glycolytic Pathway • RBC energy—glycolysis • Deficiencies in enzymes • Decreased ATP and impaired cation pumping • ↑ osmotic fragility, hemolysis

19. Glycolytic Pathway • Rapoport-Luebering shunt • Provides RBC with 2,3-bisphosphoglycerate (2,3-BPG) • Stimulated during hypoxia to facilitate O2 delivery to tissues • 2,3-BPG + Hb = Hb O2 affinity ↓ • Released by O2 to tissues

20. Clinical and Laboratory Findings • Clinical presentation variable • Ranges from no anemia to acute anemia • Normocytic/normochromic anemia • Reticulocytosis • Hyperbilirubinemia • Neonatal jaundice • Hereditary nonspherocytic hemolytic anemia (chronic)

21. Diagnosis • Diagnostic workup suggested by: • Absence of a detectable abnormal Hb • A negative direct antiglobulin test • Lack of spherocytes • Normal erythrocyte fragility test

22. Diagnosis • Definitive diagnosis • Spectrophotometric measurement • Molecular testing for specific gene • Timing of test is important • Not immediately following a hemolytic attack • Not after transfusion

23. Glucose-6-Phosphate Dehydrogenase Deficiency

24. G6PD—Introduction • First recognized during Korean War • Antimalarial drug primaquine caused HA • Worldwide • Primarily Mediterranean area, Africa, and China • X-linked inheritance—fully expressed in males and females with homozygous inheritance

25. G6PD—Introduction • Worldwide (> 400 variants) • Varying levels of severity • Generally asymptomatic except when challenged with oxidizing chemical, drug, or severe infection • May have protection against malaria

26. Table 18-2 Compounds Associated with Hemolysis in G6PD Deficiency

27. G6PD—Pathophysiology • G6PD deficiency • Generation of NADPH impaired • Generation of GSH impaired • Cellular oxidants accumulate • Hb has decreased solubility • Precipitates to form Heinz bodies • Heinz bodies attach to the RBC membrane • Supravital stain to visualize Heinz bodies continued on next slide

28. G6PD—Pathophysiology • G6PD deficiency • Heinz bodies attach to the RBC membrane: • Cause increased cation permeability, osmotic fragility, cell rigidity • Removal by splenic macrophages producing "bite" cells and blister cells • Progressive membrane loss • May form spherocytes • Extravascular hemolysis in spleen

29. Figure 18-2 Peripheral blood from a patient with G6PD deficiency during a hemolytic episode. There are erythrocytes with a portion of the cell, known as a bite cell, missing. The spleen pits the Heinz bodies with a portion of the cell producing these misshapen erythrocytes. Some of the cells reseal and become spherocytes (Wright-Giemsa stain, 1000× magnification).

30. G6PD—Pathophysiology • Oxidant stress • Oxidizes membrane lipids and proteins • Membrane damage • RBCs removed by spleen • Cells can hemolyze in circulation (intravascular hemolysis) • Hemoglobinuria • Hemoglobinemia

31. G6PD—Pathophysiology • G6PD activity • Takes very little to maintain adequate levels of GSH under normal conditions • Higher activity in young cells • Reticucolytes have 5× higher enzyme activity than oldest circulating RBCs • Reticulocytosis • G6PD deficient patients can have normal activity

32. G6PD—Pathophysiology • Severe oxidant stress • Overwhelm the system • Hemolysis is generally self-limited • Older stressed cells are hemolyzed • Replaced by younger cells with higher GSH levels

33. G6PD Variants • More than 400 variants identified • Most with normal activity • Differ in activity, stability, and electrophoretic mobility • Five classes based on degree of • Deficiency • Hemolysis

34. Table 18-3 WHO Classification of Mutant G6PD Alleles

35. Females with G6PD Deficiency • Female heterozygotes • Two populations of cells • One normal • One G6PD deficient • Lyonization • Random inactivation of one X chromosome in each cell continued on next slide

36. Females with G6PD Deficiency • Female heterozygotes • Depending on proportion of abnormal erythrocytes • No clinical expression of deficiency to severely affected

37. G6PD—Clinical Findings • Spectrum of clinical presentations • Acute acquired HA (episodic) • Favism • Congenital nonspherocytic HA—chronic • Neonatal hyperbilirubinemia with jaundice • Homozygotes and heterozygotes • Symptomatic depending on severity of deficiency

38. G6PD—Clinical Findings • Acute, acquired hemolytic anemia • Most have no clinical symptoms, no anemia • Hemolytic episodes occur after • Infectious illness • Exposure to certain drugs continued on next slide

39. G6PD—Clinical Findings • Acute, acquired hemolytic anemia • Variable hemolysis, depends on • Degree of oxidant stress • G6PD variant • Sex of patient continued on next slide

40. G6PD—Clinical Findings • Acute, acquired hemolytic anemia • Drug-induced • Acute intravascular hemolysis • 1–3 days after exposure • 3–4 g/dL drop in Hb • Abdominal and lower back pain • Dark or black urine continued on next slide

41. G6PD—Clinical Findings • Acute, acquired hemolytic anemia • Ingestion of fava beans (broad beans) • Sudden severe hemolytic episode (favism) • Similar to drug-induced episodes • Variants involved • G6PD Mediterranean variant, G6PD-A-, G6PD Aures • Usually affects children 2–5 years old

42. G6PD—Clinical Findings • Findings • Malaise, severe lethargy, nausea, vomiting, abdominal pain, chills, tremor, fever • Hemoglobinuria a few hrs after ingestion • Jaundice

43. G6PD—Clinical Findings • Hereditary (chronic) nonspherocytic HA • Associated with G6PD variants (Class I) • Low in vitro activity or are markedly unstable • Hemolysis • Chronic • Not associated with ingestion of drugs or infections, although they can exaggerate the hemolysis continued on next slide

44. G6PD—Clinical Findings • Hereditary (chronic) nonspherocytic HA • Associated with G6PD variants (Class I) • Hemolysis usually compensated, so mild anemia • Reticulocytosis: 4–35%

45. G6PD—Clinical Findings • Neonatal hyperbilirubinemia • Some neonates with G6PD deficiency • Severe hyperbilirubinemia • Potential for kernicterus • Exchange transfusion and phototherapy

46. G6PD—Laboratory Findings • Most variants • No anemia and normal blood findings until hemolytic episode

47. Laboratory Findings • Immediately following hemolytic episode • Peripheral blood • Polychromasia, occasional spherocytes, small hypochromic cells, RBC fragments, bite cells • Blister cells continued on next slide

48. Laboratory Findings • Immediately following hemolytic episode • Peripheral blood • Reticulocytosis • ↑ Leukocytes • ↑ unconjugated bilirubin and serum LD • ↓ haptoglobin

49. Figure 18-3 The arrows are pointing to blister cells, which have various names (see the chapter text). Hemoglobin is condensed to one side of the cell, leaving a transparent area (blister) on the other. These cells can be found in G6PD-deficient individuals after a hemolytic attack (Wright-Giemsa stain, 1000 magnification).

50. G6PD—Laboratory Findings • Definitive diagnosis • Requires demonstration of a ↓ in erythrocytic G6PD activity • Timing is critical • Perform assays 2–3 months after hemolytic episode