This document discusses hemolytic anemias, including hereditary spherocytosis, G6PD deficiency, and paroxysmal nocturnal hemoglobinuria (PNH). Hereditary spherocytosis is caused by defects in membrane cytoskeletal proteins that result in spherical and fragile red blood cells prone to splenic sequestration and destruction. G6PD deficiency leaves red blood cells vulnerable to oxidative stress from drugs or infections. PNH is an acquired disorder where mutations lead to deficiencies in proteins that regulate complement activity on red blood cells, making them susceptible to complement-mediated hemolysis, especially at night.

1. HEMOLYTIC ANEMIAS
Dr.CSBR.Prasad, M.D.,

2. Clinical presentation
• Anemia
• Jaundice
• Organomegaly
• Delayed mile stones
• Dysmorphic facies
• Gall stones in young

3. Hemolytic anemias
Common features:
• Premature destruction of red cells
• Elevated erythropoietin
• Accumulation of hemoglobin degradation
products

4. Destruction of RBCs
• Physiologically takes place in mononuclear
phagocytic system (Spleen,Liver, BM)
• Terms:
– Extravascular hemolysis
– Intravascular hemolysis

5. Extravascular hemolysis
• Caused by alterations that render the red cell
less deformable
• Clinical features:
– Anemia
– Splenomegaly &
– Jaundice

6. Intravascular hemolysis
Causes:
• Mechanical injury
– Prosthetic heart valves
– Fibrin Thrombi in microcirculation or
– Repetitive physical trauma
• Marathon running
• Complement fixation
– Abs to RBCs
• Intracellular parasites
– Malaria
• Exogenous toxic factors
– Clostridial sepsis

7. Intravascular hemolysis
Manifested by:
• Anemia
• Hemoglobinemia
• Hemoglobinuria
• Hemosiderinuria, and
• Jaundice
• No splenomegaly

8. Intravascular hemolysis
• Low serum haptoglobin
• Methemoglobinemia
• Renal hemosiderosis
• Elevated serum bilirubin levels (UC)
• Gall stones
• Increased urobilinogen

9. Hemolytic Anemias - investigations
• Peripheral smear
– Fragmented RBC, nucleated RBC, polychromatophilic cells,
• ↑ Reticulocytes
• Bone marrow
– ↑ Cellularity, M:E ratio 1:1 (Erythroid hyperplasia)
– ↑ Normoblasts
• Extramedullary hematopoiesis
• ↑ Bilirubin – cholelithiasis
• Hemosiderosis

10. Red cell sequestration in splenic sinusoids

11. There are numerous fragmented RBC's seen here. Some of the irregular shapes appear
as "helmet" cells. Such fragmented RBC's are known as "schistocytes" and they are
indicative of a microangiopathic hemolytic anemia (MAHA) or other cause for
intravascular hemolysis. This finding is typical for disseminated intravascular
coagulopathy (DIC).

12. Polychromatophilic cells

13. Reticulocyte

14. Normal marrow fragments and cell trails.
Fig. 1.24

15. Hereditary Spherocytosis (HS)

16. • Inheritance
– AD (75%)
– Compound heterozygosity
• Northern Europe (1 in 5000)
• Defective cell membrane skeleton
• Spherical & less deformable RBC
• Splenic sequestration % destruction

17. Pathogenesis
• Cytoskeletal defect
• The life span of the affected red cells 10 to 20
days

18. Hereditary Spherocytosis
• Spectrin – Major protein of membrane
cytoskeleton
• Two polypeptide chains – α and β
• Spectrin is tethered to the inner surface of cell
membrane by ankyrin, protein 4.2 to trans
membrane transporter band 3

21. Hereditary Spherocytosis
• Gene mutation involving ankyrin, protein 4.2,
spectrin or band 3 reduce membrane stability
• Spontaneous loss of cell membrane
• ↓ Cell surface to volume ratio – spheroidal shape
• Spherocytes are less deformable and vulnerable to
splenic sequestration and destruction

22. Schematic representation of the red cell membrane cytoskeleton
and alterations leading to spherocytosis and hemolysis

23. Red cell sequestration in splenic sinusoids

24. Hereditary Spherocytosis
• Clinical features
– Anemia - varies
– Splenomegaly
– Jaundice
• Aplastic crisis – triggered by parvovirus
infection of marrow precursor
• Hemolytic crisis

25. Hereditary Spherocytosis
• Hb – normal / decreased
• MCV – decreased
• MCHC – increased more than 36 gm/dl
• PBS
– Spherocytes
– Reticulocytosis – more than 8%
– Nucleated RBC
• Osmotic fragility test

26. Osmotic fragility test

27. Osmotic fragility test

28. Hereditary Spherocytosis

29. Hereditory Spherocytosis

30. The size of many of these RBC's is quite small, with lack of the central zone of pallor.
These RBC's are spherocytes. In hereditary spherocytosis, there is a lack of spectrin, a
key RBC cytoskeletal membrane protein. This produces membrane instability that
forces the cell to the smallest volume--a sphere. In the laboratory, this is shown by
increased osmotic fragility. The spherocytes do not survive as long as normal RBC's.

31. Reticulin stain

32. Hereditary Spherocytosis
• Osmotic fragility test – Confirmatory test
• Spherocytes are vulnerable to osmotic lysis
induced in vitro by hypotonic salt solution
• Hemolysis starts at 0.8 gm% and completes
between 0.5 – 0.4 gm%

33. G6PD - deficiency

34. Name the two important products of
HMP shunt
• NADPH
• Ribose-5-Phophate

36. G6PD - deficiency
• Erythrocytes are vulnerable to oxidant
induced injury
• Intracellular reduced glutathione (GSH)
inactivates oxidant
• G6PD is needed for maintaining adequate
quantity of GSH

37. G6PD - deficiency
• Sulfhydral group of globin chain of Hb is
oxidized.
• Hb precipitate to form Heinz bodies – damage
the cell membrane
• Bite cells
• Intravascular hemolysis

39. Heinz bodies

40. G6PD - deficiency
Oxidant stress
• Drugs
– Antimalaria – primaquine
– Sulfonamide
– Sulfones
– Nitrofurans
– Analgesic
• Infection – Viral hepatitis, pneumonia, typhoid fever
• Food – fava beans (Favism)

41. G6PD - deficiency
• X – linked disorder
• More than 350 G6PD genetic variants are recognised
• G6PD A-
– 10% of American black
– Normal enzyme activity in reticulocyte
– Unstable enzyme – half time 13 days (62 days)
• G6PD Mediterranean
– Severe ↓ enzyme activity – less than 10%
– Severe hemolysis

42. G6PD - deficiency
• Asymptomatic
• Infectious disease / drug exposure
• Sudden onset of anemia
• Hemoglobinuria, Hemoglobinemia
• Abdominal / low back pain
• Self limited

43. G6PD - deficiency
• PBS
– Spherocytes, erythrocyte fragments, bite cells,
Heinz bodies, polychromasia
• Measurement of enzyme activity
– Fluorescent spot test
– Dye reduction test

44. Polychromatophilic cells

45. PNH
Paroxysmal Nocturnal Hemoglobinuria

46. • Incidence of 2 to 5 per million

47. Acquired Membrane disorder
• Paroxysmal nocturnal hemoglobinuria (PNH)
is a disease that results from acquired
mutations in the phosphatidylinositol glycan
complementation group A gene (PIGA), an
enzyme that is essential for the synthesis of
certain cell surface proteins

48. Attachment of proteins to cell
membrane
• Transmembrane proteins
• GPI linked proteins: The others are attached
to the cell membrane through a covalent
linkage to a specialized phospholipid called
glycosylphosphatidylinositol (GPI).
• In PNH, these GPI-linked proteins are
deficient because of somatic mutations that
inactivate PIGA

49. • PNH blood cells are deficient in three GPI-linked
proteins that regulate complement activity:
1. decay–accelerating factor, or CD55
2. Membrane inhibitor of reactive lysis, or CD59
3. C8 binding protein.
• Of these factors, the most important is CD59, a
potent inhibitor of C3 convertase that prevents
the spontaneous activation of the alternative
complement pathway.

50. • Red cells, platelets, and granulocytes
deficient in these GPI-linked factors are
abnormally susceptible to lysis or injury by
complement
• In red cells this manifests as intravascular
hemolysis
• The hemolysis is paroxysmal and nocturnal

51. Urine samples from a
typical PNH patient

52. PNH is diagnosed by flow cytometry

53. Major vein thrombosis
• Thrombosis is the leading cause of disease-
related death in individuals with PNH
• Other causes:
– Protein C / S deficiency
– Factor V Leiden
– Hyperfibrinogenemia

54. END

55. Dr.CSBR.Prasad, M.D.,
Associate Professor of Pathology,
Sri Devaraj Urs Medical College,
Kolar-563101,
Karnataka,
INDIA.
csbrprasad@reiffmail.com