1. Advanced Hematology (Medi 6304)
HEMOLYTIC
ANMEIA
Ahmad Silmi
Second semester
MAR 6th 2017

2. Hemolytic anemia: Definition
Anemia results from an excessive hemolysis of RBC’s
BM compensatory capacity: 6-8 folds of normal
 Several folds destruction rate
 Life span with erythrocytes less than 30 days
 Anemia developed (Signs & symptoms)
Two type of hemolysis:
1. Intra-vascular hemolysis
2. Extra-vascular hemolysis

3. Hemolytic anemia: Hemolysis routes
Intravascular Hemolysis
Senescence: Reduced biological activities
 Glycolytic pathway deterioration
- Loss of NADPH and NADH pool
- loss of ATP generation:
 loss of Membrane permeability & deformability
 Structural membrane abnormalities
 Intracelluar Na+ () Intracelluar K+ ()
 Surface-volume ratio ()  Spheroidals h

4. Hemolytic anemia: Hemolysis routes
Intravascular Hemolysis (cont.)
Hemoglobin release into the circulation (Free)
Loaded on Haptoglobin (To BM or Liver)
Upon haptoglobin saturation
- Free Hemoglobin filtration (Kidney) & Reabsorption
- Excessive hemolysis
 Reabsorption capacity exceeded
 Hemoglobinuria and Hemoglobinemia

5. Hemolytic anemia: Hemolysis routes
Extravascular Hemolysis (cont.)
IgG accumulation on effete erythrocytes
RES Macrophages recognition & phagocytosis
Degradative enzymatic digestion by macrophages
Hemoglobin disassembling:
- Iron released  Transferrin  BM or Hepatocytes
- Globin  components amino acids  a.a. pool
- Protoporphyrin  Bilirubin  Conjugated (Liver)
 Intestine (via Bile)
 Stercobilinogen & Stercobilin (with feces)
 Reabsorption  to Kidney  Urobilinogen (Urine

6. Hemolytic anemia: Hemolysis routes

7. Bilirubin

8. Bilirubin

9. Bilirubin

10. Bilirubin

11. Hemolytic anemia: Clinical and diagnostic features
Associated with excessive destruction of erythrocytes
 Liver capacity fail to conjugate
 Bilirubin concentration elevated
 Jaundice
 Urobilinogen (in urine) elevated
Darkening of urine on standing
 Stercobilinogen (in feces) elevated
 Serum haptoglobin: absent

12. Hemolytic anemia: Complications
Hyperbilirubinemia

13. Hemolytic anemia: Complications
Hyperbilirubinemia

14. Hemolytic anemia: Complications
Hyperbilirubinemia

15. Hemolytic anemia: Complications
Hypercoagulability

16. Hemolytic anemia: Complications
Hypercoagulability

17. Hemolytic anemia: Complications
Hypercoagulability

18. Hemolytic anemia: Complications
Hypercoagulability

19. Hemolytic anemia: Clinical and diagnostic features
CBC and Blood film examination

20. Hemolytic anemia: Clinical features

21. Hemolytic anemia:
Diagnostic features

22. Hemolytic anemia: Clinical and diagnostic features
CBC and Blood film examination

23. Hemolytic anemia: Clinical and diagnostic features
CBC and Blood film examination

24. Hemolytic anemia: Clinical and diagnostic features
Associated with increased erythropoiesis rate:
 Reticulocytosis and other evidences of BM hyperactivation

25. Hemolytic anemia: Clinical and diagnostic features
Associated with increased erythropoiesis rate:
 Reticulocytosis and other evidences of BM hyperactivation

26. Hemolytic anemia: Clinical and diagnostic features
Associated with increased erythropoiesis rate:
 Reticulocytosis and other evidences of BM hyperactivation

27. Hemolytic anemia: Clinical and diagnostic features
Associated with increased erythropoiesis rate:
 BM index (Hyperplasia): Myeloid:Erthroid Hyperplasia (1:1)

28. Hemolytic anemia: Clinical and diagnostic features
Associated with morphological abnormalities:
 Osmotic fragility and Autohemolysis
- Incubation of test RBC’s in hypotonic solution under sterile
conditions for 24 hours.

29. Hemolytic anemia: classification
Classification: Based on the etiology and pathophysiology
1. Hereditary: Acquired:
a. Membrane defects a. Immune-mediated:
(ex. Spherocytosis) Auto~ , Allo~ , Drug-induced ~
b. Metabolic defects b. Microangipathic (MAHA)
(ex. G6PD deficiency) TTP, HUS, DIC
c. Haemoglobin Abnormalities c. Paroxysmal Noctunal Haemoglobinuria
(ex. Hb-S and Hb-C) d. Secondary:
Infections, Liver and renal disease

30. Hemolytic Anemia: Membrane defect
> Vertical interactions stabilize the lipid bilayer.
>> Deficiencies of spectrin, ankyrin, or band 3 protein causes decoupling of the lipid bilayer from the underlying skeletal lattice
>>>subsequent membrane loss in the form of microvesicles.
>>>>This leads to the formation of spherocytes (hereditary spherocytosis).

31. Hemolytic Anemia: Membrane defect
> Horizontal interactions provide mechanical stability to the membrane.
>> defects include abnormal spectrin heterodimer association to form tetramers and defective skeletal protein interactions of junctional complexes at the end of the spectrin tetramers (spectrin, actin, protein 4.1)
>>> result in fragmentation of the red blood cell (schistocytosis).

32. Hereditary spherocytosis
HS Molecular Defects: Spectrin
- Spectrin deficiency is the most common protein alteration in HS.
- Not all spectrin defects lead to the formation of spherocytes.
- Mutations that affect the self-association of Spectrin heterodimers lead to hereditary elliptocytosis.
HS Molecular Defects: Ankyrin
- Patients with ankyrin defects have prominent spherocytosis without other morphologic defects.
- About 2/3 of patients have a combined defect of both ankyrin and spectrin.

33. Hereditary spherocytosis
HS Molecular Defects: Band 3
- Generally milder.
- Many of the mutations affect the protein’s ability to be inserted into membranes
- All patients with band 3 deficiency have a proportional
decrease in protein 4.2 because band 3 is apparently needed for 4.2 stability.
HS Molecular Defects: Band 4.2
- Protein 4.2 deficiency is associated with loss of band 3; and, in some cases, a loss of ankyrin.
- The morphology in these patients is variable and sometimes stomatocytes and ovalocytes are more common than spherocytes.

35. Hereditary spherocytosis: Diagnosis
Required criteria:
At least one sign of increased red cell destruction:
(ex. reticulocytosis, increased bilirubin, decreased haptoglobin, increased lactate dehydrogenase).
Increased spherocytes and anisocytosis on the blood film.
Increased red cell osmotic fragility.
Optional criteria
1. Positive family history
2. Splenomegaly
3. Anemia (1/3 of HS patients are NOT anemic)

36. Hereditary ellipocytosis:
- HE is inherited as an autosomal dominant gene. Heterozygous individuals are asymptomatic; homozygous individuals have mild to severe
hemolysis.
- Spectrin and band 4.1 mutations are invloved in the etiology of HE; abnormalities in protein 4.1 are much less common than spectrin mutations

37. Hereditary ellipocytosis:
- The hallmark is the presence of large numbers of elliptocytes in the peripheral blood.
- Red cell fragmentation may be seen in the more severe forms.
- Osmotic fragility is generally normal.
- If hemolysis is present, reticulocytosis, and other evidence of red cell destruction will be apparent as listed earlier for HS.

38. Hemolytic anemia: classification
Classification: Based on the etiology and pathophysiology
1. Hereditary: Acquired:
a. Membrane defects a. Immune-mediated:
(ex. Ellipocytosis, Spherocytosis) Auto~ , Allo~ , Drug-induced ~
b. Metabolic defects b. Microangipathic (MAHA)
(ex. G6PD deficiency) TTP, HUS, DIC
c. Haemoglobin Abnormalities c. Paroxysmal Noctunal Haemoglobinuria
(ex. Hb-S and Hb-C) d. Secondary:
Infections, Liver and renal disease

39. Glucose 6-phosphate dehydrogenase (G6PD):
Glucose G6P PG
NADP NADPH
Hexose
Monophosphate
Shunt
Reductase
GSH GSSH
Peroxidase
H H2O

40. G6PD deficiency:

41. G6PD deficiency:

42. G6PD deficiency: G6PD gene is located on the X chromosome (q28).
Lyon’s hypothesis:
Heterozygous female: 2 cell populations
50% of cell are G6PD normal & 50% are deficient
Clinically: features ranging from normal to severely deficient
G6PD variants:
- Point mutations (single amino acid substitution)
- >400 G6PD variants were identified.
- variants shows variable enzyme stability and/or kinetics

43. G6PD variants: Class G6PD activity (% normal) Clinical outcomes Ex. I
< 10%
chronic nonspherocytic hemolytic anemia (CNSHA) II
No CNSHA but can lead to severe and episodic acute hemolytic anemia.
G6PD-Med
III
10% - 60%
No CNSHA but can lead to severe and episodic acute hemolytic anemia that usually is self-limiting
G6PD-A– IV
60% - 150%
associated with no hemolysis
G6PD-B V
>150%
Not clinically relevant
---

44. Chronic nonspherocytic hemolytic anemia (CNHA)
Evidenced by chronic hyperbilirubinemia, decreased serum haptoglobin level, and increased lactate dehydrogenase level.
The RBC morphology is unremarkable and is referred to as nonspherocytic.
The hemolysis is mostly extravascular (no hemoglobinurea).
Characterized by chronic anemia diagnosed at birth as having neonatal hyperbilirubinemia and the hemolysis continues into adulthood.
Usually with compensatory reticulocytosis but can be transfusion dependent.

45. G6PD deficiency: Clinical features
Normally, patients are asymptomatic till ….
Hemolytic crises: Intravascular hemolytic anemia following to the exposure of oxidizing agents
jaundice: 2-3 days after exposure
Anemia worsens till the 7th-8th day
Self limiting anemia
G6PD deficiency & Favism
G6PD deficiency and Resistance to P. falciparum

46. G6PD deficiency
Pathogenesis

47. G6PD deficiency: Consequences
Blister cells:
red cells with a clearing at the periphery, like a blister
Arises when bite cells undergo repair of the cell membrane, resulting in a clearing within the red cell where the Heinz body previous was.
Bite cells: red cells with ‘bites’ taken out
Bites’ are from splenic macrophages removing the part of the red blood cell with a Heinz body.

48. G6PD deficiency: diagnosis

49. G6PD deficiency: Following to oxidative stress
Blood film morphology:
Normocytic, normochromic anemia
Red cells fragmentation ( Bite cells and Blister cells)
Occasional spherocytes
Dimorphisms (Females)

50. G6PD deficiency: Following to oxidative stress
Hemolytic features:
Increased reticulocytes after hemolytic crises
Hemoglobinemia & Hemoglobinuria

51. G6PD deficiency: Following to oxidative stress
Heinz bodies:
Inclusions of denatured hemoglobin (methemoglobin) precipitate
Satin: Supravital brilliant crysel blue

52. Acquired Hemolytic Anemia
Acquired immune-mediated

53. Hemolytic anemia: classification
Classification: Based on the etiology and pathophysiology
1. Hereditary: Acquired:
a. Membrane defects a. Immune-mediated:
(ex. Ellipocytosis, Spherocytosis) Auto~ , Allo~ , Drug-induced ~
b. Metabolic defects b. Microangipathic (MAHA)
(ex. G6PD deficiency) TTP, HUS, DIC
c. Haemoglobin Abnormalities c. Paroxysmal Nocturnal Haemoglobinuria
(ex. Hb-S and Hb-C) d. Secondary:
Infections, Liver and renal disease

54. Immune-mediated hemolytic anemia
Is the shortening of the red cell’s life span due to production of antibodies against one or more antigens on the surface of the red cell.
Immune Hemolytic Anemia may be either
1. Allo-immune: Antibodies generated against foreign (non-self) antigens
2. Auto-immune : Antibodies generated against self (auto) antigens

55. Allo-immune hemolytic anemia
mediated by antibodies generated against foreign (non-self) antigens
- Blood transfusion
Hemolytic transfusion reaction (ABO/Rh incompatibility)
- Stem cell transplantation
Passenger Lymphocyte Syndrome (minor ABO incopatibility)
- Pregnancy
Hemolytic disease of newborn (mother’s Ab’s against fetus Ag’s)

56. Hemolytic disease of newborn

57. Auto-immune hemolytic anemia
Warm autoimmune H.A.
Represent approximately 75% of all AIHA cases
Antibodies:
- Reactivity: Rh and Kell antigens
- Class: IgG (with or without complement)
- Optimal reactivity: at 37oC
Primary (30%) and secondary (70%)
Methyldopa therapy or Evans’ syndrome (idiopathic-thrombocytopenic purpura)
Hemolysis is a result of adherence of IgG-Fc fragment on sensitized RBCs to their receptors on macrophages and subsequent phagocytosis extravascular hemolysis)

58. Autoimmune hemolytic anemia
Warm autoimmune H.A.
Laboratory Findings: (Typical features of extra-vascular hemolysis)
- Red cells may be macrocytic due to large numbers of reticulocytes
- Marked anisocytosis with spherocytosis (hemolysis)

59. Autoimmune hemolytic anemia
Warm autoimmune H.A.
Laboratory Findings:
- Direct antibody test (DAT) is positive in the majority of cases.
- The indirect antiglobulin test may demonstrate Autoantibodies

60. Coombs’ test

61. Auto-immune hemolytic anemia
Cold-agglutinins autoimmune H.A.
Antibodies: - Reactivity: Ii blood grouping antigens
- Class: Usually IgM (occasionaly IgG)
- Optimal reactivity: at 0- 4 Oc
- Ab’s are capable of binding complement
Hemolysis is usually extra-vascular in the liver due to the C3b receptors on Kupffer cells.
When the patient is exposed to cold, agglutination of red cells in the small capillaries causes numbness, pain, and skin discoloration (acrocyanosis or Reynaud’s phenomenon). Gangrene may develop in severe cases.

62. Cold- agglutinins hemolytic anemia
Laboratory Findings:
- Hemoglobin may fluctuate with seasons.
- Degree of reticulocytosis and bilirubin concentration varies with severity of red cell hemolysis.
- Clumping of red cells at room temperature may lead to erroneous laboratory data from automated instruments.
- For patients experiencing hemolysis, the cold agglutinin titer at 4oC is over 1000 (it is generally under 64 in healthy individuals).
- The DAT is usually positive when a polyspecific antiglobulin serum is used that detects both complement and antibodies on the red cell surface.

63. Paroxysmal nocturnal hemoglobinuria
Defective synthesis of GPI (X-chromosome mutation)
Lack of GPI anchored proteins (DAF/CD55 and MIRL/CD59)
RBC’s are susceptible to complement mediated lysis
Intravascular hemolysis

64. Paroxysmal nocturnal hemoglobinuria
Clinical features
People with PNH may have underlying bone marrow hypoplasia
Wide variation of clinical features ( frequency and severity)