Hemolytic anemia

1. Hemolytic Anemia

2. Hemolytic Anemia
Hemolysis is premature destruction of erythrocytes
It leads to anemia when the bone marrow activity can
not compensate for the erythrocyte loss.
Bone marrow has the capacity to increase the RBC
production by 6 to 8 folds. Thus, shortening of
erythrocyte life span to a corresponding degree
remains compensated and anemia does not manifest
(Hemolytic disorder)

3. Hemolytic Anemia
When shortening of red cell life span is beyond the
regenerative capacity of the bone marrow Hemolytic
anemia manifests.
Represent 5% of all anemias

4. HEMOLYTIC ANEMIA
Reduced life span of RBC (normal 120 days)
Accumulation of products of Hb catabolism
Increased erythropoiesis in the marrow and other
sites (leading to reticulocytosis)
Clinical triad:
Anemia
Jaundice
Splenomegaly

5. Classification of Hemolytic Anemia
Intrinsic (intracorpuscular abnormality of red cells)
Hereditary
• Red cell membrane disorders
 Disorders of membrane cytoskeleton: spherocytosis, elliptocytosis
 Disorders of lipid synthesis : selective increase in membrane lecithin
• Red cell enzyme deficiencies
Glycolytic enzymes: pyruvate kinase, hexokinase deficiency.
Enzymes of hexose monophosphate shunt: glucose 6 phosphate
dehydrogenase, glutathione synthetase.
• Disorders of hemoglobin synthesis
Deficient globin synthesis : thalassemia syndromes
Structurally abnormal globin synthesis(hemoglobinopathies): sickle cell
anemia
Acquired
• Paroxysmal nocturnal hemoglobinuria

6. Classification of Hemolytic Anemia
Extrinsic (extracorpuscular abnormalities)
• Antibody mediated
Isohemagglutinins: transfusion reactions, erythroblastosis
fetalis
Autoantibodies: idiopathic(primary), drug associated,
systemic lupus erythematosus, malignant neoplasms,
mycoplasma infection.
• Mechanical trauma to red cells
Microangiopathic hemolytic anemia : thrombotic
thrombocytopenia purpura, disseminated intravascular
coagulation.
Cardiac traumatic hemolytic anemia
• Infections : malaria, hookworm, Clostridium welchii
• Sequestration in mononuclear phagocyte system: hypersplenism
• Chemical injury: lead poisoning

7. Normal physiology of Hb metabolism

8. Hemolysis can be:
•Intravascular
•Extravascular

9. Intravascular Hemolysis
Lysis of red cells within the vascular compartment.
 It may be caused by mechanical injury, complement fixation,
infection or exogenous toxins.
The Hb released binds to haptoglobin and hemopexin leading
to decresed serum haptoglobin and hemopexin
When plasma Hb exceeds the haptoglobin binding capacity Hb
is excreted in urine (hemoglobinuria)
Hemosiderinuria occurs in chronic intravascular hemolysis.

10. Extravascular hemolysis :
Takes place whenever the red cells are rendered
foreign or become less deformable .
Since extreme alteration in shape is required for the
red cells to navigate the splenic sinusoids reduced
deformability makes the passage difficult leading to
sequestration and phagocytosis
Work hyperplasia of the spleen leads to
splenomegaly

11. TEST INTRAVASCULAR EXTRAVASCULAR
Reticulocyte
count
LDH
Indirect Bilirubin
Hemoglobinuria + -
Hemosiderinuria + -
Splenomegaly mild mod to severe

12. Approach to diagnosis
• To establish the presence of hemolysis
• To determine the type of hemolysis (intravascular or extravascular)
• To find out the underlying cause of hemolysis

13. When to suspect hemolytic anemia?
• Clinical manifestations
Pallor
Jaundice
Splenomegaly
Gallstones
Skeletal abnormalities
Leg ulcers

14. When to suspect hemolytic anemia (contd…)
 PBS shows :anisopoikilocytosis, macrocytes,
polychromasia, schistocytes, spherocytes,
elliptocytes, target cells, sickle cells, basophilic
stippling.
 Reticulocytosis
 High: e.g. thalassemia, sickle cell anemia
 Very high e.g. hereditary spherocytosis, elliptocytosis.

15. Spherocytes
• Hereditary spherocytosis
• Immunohemolytic anemia
• Burns

16. Elliptocytes

17. Schistocytes

18. Target cells
• Thalassemia
• Sickle cell anemia
• Liver disease
• Hb C disorders

19. Acanthocytes
• Liver disease
• Abetalipoproteinemia

20. Echinocytes

23. Investigations to establish accelerated
hemolysis
Increase in the catabolic products of hemoglobin in
the blood
1. Unconjugated hyperbilirubinemia
2. Hemoglobinemia (intravascular hemolysis)
3. Hemoglobinuria (intravascular hemolysis)
4. Hemosiderinuria (intravascular hemolysis)
5. Increased urinary urobilinogen
 Effects due to release of hemoglobin
1. Depletion of serum haptoglobin
2. Depletion of serum hemopexin
 Increase in serum LDH

24. Red Cell Membrane Abnormalities
• Occur due to defect in
Membrane cytoskeleton
• Spherocytosis
• Elliptocytosis
Lipid synthesis
• Selective increase in membrane lecithin

25. Hereditary Spherocytosis
• Autosomal dominant pattern of inheritance
• HS is caused by mutations affecting ankyrin(most common), band3,
spectrin or band 4.2
• Reduced membrane stability leads to loss of membrane fragments
during exposure to shear stress in the circulation
• The loss of membrane relative to cytoplasm forces the cell to assume
the smallest possible diameter for a given volume i.e. sphere

26. Hereditary Spherocytosis
•The spherocytes can not undergo extreme
deformation while traversing the splenic cords
leading to stagnation and ultimately phagocytosis
by the macrophages

27. When to suspect HS?
• Hemolytic facies and skeletal abnormalities
• Positive family history
• Presence of spherocytes in peripheral blood smear

28. PBS showing spherocytes

29. Diagnosis of membrane defects
Osmotic fragility test :
 Blood is mixed with saline solutions of varying concentration. The
fraction of red cells lysed is determined colorimetrically
 Shift of curve to right with tailing at end is highly suggestive of
hereditary spherocytosis. Changes are less marked in elliptocytosis

30. Approach to diagnosis of membrane defects
(contd…..)
• Autohemolysis : Normal autohemolysis virtually rules out membrane
and enzyme abnormalities. Partial correction of increased
autohemolysis by glucose is highly suggestive of red cell membrane
defect
• Family screening to establish the hereditary nature
• Cell membrane protein analysis by SDS -PAGE

31. PNH
• Membrane proteins are anchored to the lipid bilayer in 2 ways
Hydrophobic proteins span the red cell
membrane(transmembrane proteins)
Attached to the membrane by a specialised phospholipid
called GPI
• PNH results from acquired mutations in phosphtidyl inositol
glycan A (PIGA), which is essential for the synthesis of GPI
anchor
• PIGA is X linked

32. PNH (contd…)
• The causative somatic mutations occur in the pluripotent stem
cells, hence all its clonal progeny (RBC, WBC & platelets) are
deficient in proteins attached to the cell membrane via GPI
• Several GPI linked proteins inactivate complement & their
absence in PNH renders the blood cells unusually sensitive to
lysis by complement
• Not all the blood cells are affected in PNH patients indicating
that the mutant clone exists along with the normal stem cells

33. PNH
• Three GPI linked proteins regulate complement activity
Decay accelerating factor or CD55
Membrane inhibitor of reactive lysis or CD 59
( potent inhibitor of C3 convertase thereby preventing
spontaneous activation of alternate complement pathway )
C 8 binding protein

34. When to suspect acquired membrane
abnormality- PNH?
• Episodic nocturnal hemoglobinuria
• Recurrent iron deficiency anemia (due to hemosiderinuria)
• Pancytopenia with absent to mild splenomegaly
• Episodic venous thrombosis (platelet dysfunction)
• Chronic hemolytic anemia with other causes of hemolysis
ruled out

35. To confirm the diagnosis of PNH
• Establish intravascular hemolysis
• Establish increased sensitivity of red cells to complement
mediated lysis
Acidified serum test/ Ham test
Sucrose lysis test
• Demonstrate deficiency of CD 55& or CD59 in patients RBCs
and neutrophils by flow cytometry

36. Hemolytic anemia due to hemoglobin
abnormality
•Quantitative reduction of globin chain synthesis
known as thalassemia syndromes
•Qualitative / structural defect in globin chain leading
to hemoglobinopathies
•Hereditary persistence of foetal hemoglobin

37. Thalassemia syndromes
• Decreased synthesis of either α or β globin chain of Hb A
• Hematological consequences result from
Low intracellular Hb (hypochromia)
In β thalassemia the excess of α chains aggregate into
insoluble inclusions within the red cells and their
precursors leading to premature destruction of maturing
erythroblasts in the marrow (ineffective erythropoiesis)
and lysis of mature red cells in the spleen (hemolysis)

38. β thalassemia
• Classified into 2 categories:
 β0 thalassemia associated with total absence of β globin
chain
 β+ thalassemia characterised by reduced β globin chain
synthesis
• Occur due to mutations in the β globin gene on
chromosome 11
Promoter region mutations
Chain terminator mutations
Splicing mutations

39. Clinical syndromes associated with β
thalassemia
• β thalassemia major: Homozygous for β thalassemia
genes(β+/ β+ or β0/ β0). They present with severe transfusion
dependent anemia.
• β thalassemia minor or β thalassemia trait: Heterozygotes
with one normal gene(β+/β or β0/β)
- Usually have mild microcytic anemia that causes
no symptoms.
• β thalassemia intermedia : includes milder variants of β+/
β+ or β+/ β0

40. α Thalassemia
• Occur due to mutation in the α globin gene on chromosome
16
• Clinical syndromes associated with α thalassemia
Silent carrier state: Occurs if a single α-globin gene is
deleted. There is barely detectable reduction in α –globin
chain synthesis that does not result in anemia.
α thalassemia trait: Caused by deletion of 2 α globin genes
which may be from the same chromosomes
(αα/_ _ )or from 2 chromosomes (α/_/ α/_) is a carrier
state with no anemia and no symptoms. There is
microcytosis but no physical signs of anemia

41. α Thalassemia
Hb H disease:
Caused by deletion of 3 α-globin genes.
Tetramers of excess β-globin called Hb H form.
 Hb H has extremely high affinity for O2 and is not useful
for O2 exchange leading to tissue hypoxia
disproportionate to the level of Hb.
Hb H is prone to oxidation and forms precipitates which
are stained with vital dyes.
Produces moderately severe anemia

42. α Thalassemia
• Hydrops fetalis:
Caused by deletion of all 4 α-globin genes.
In the fetus excess γ-globin chains form
tetramers(Hb Barts) with such high affinity for O2
that they deliver almost no O2 to tissues.
 The fetus shows severe pallor, generalised
edema & hepatosplenomegaly.

43. When to suspect thalassemia?
•Clinical presentation:
Age : 6-9 months after birth(β thalassemia)
Growth retardation
Prominent cheek bones and enlarged bony
prominences
Hepatosplenomegaly
Cardiac diseases

44. Thalassemia (contd…)
•PBS :
Marked anisocytosis and poikilocytosis
Microcytosis and hypochromia
Target cells
Basophilic stippling
Fragmented red cells
Normoblasts
•Reticulocytosis
 X Ray of skull shows crew cut appearance

45. β Thalassemia minor

46. β Thalassemia Major

47. To establish the diagnosis of thalassemia
• Documentation of hypochromic microcytic cells with exclusion of iron
deficiency state
• Hb electrophoresis
• NESTROFT
• Hb A2 and Hb F estimation
β thal trait: Hb A2 ≥ 4%, Hb F normal or mildly
elevated
β thal major: Hb A2 reduced or normal, Hb F
elevated
• DNA analysis and PCR to look for gene deletions
• Family studies

48. Sickle cell anemia
• Occurs due to point mutation in the β globin chain leading to
substitution of valine for glutamic acid at the sixth position
• When deoxygenated, Hb S molecules undergo aggregation
and polymerization
• Aggregated Hb S molecules assemble into long needle like
fibres within red cells producing distorted sickle shape
• Sickled red cells have rigid non deformable cell membrane
that leads to difficulty in negotiating the splenic sinusoids,
sequestration & phagocytosis

49. Sickle cell anemia (contd…)
•Reversibly sickled red cells express higher than
normal number of adhesion molecules leading to
their arrest in the microvasculature particularly in
areas of sluggish flow & in inflamed tissues
•This results in extended exposure to low oxygen
tension, causing sickling and microvascular
occlusion

50. When to suspect sickle cell anemia?
•Clinical presentation
Severe anemia
Jaundice
Vaso-occlusive complications like painful bone
crisis, acute chest syndrome
Recurrent infections
•PBS : anisocytosis, poikilocytosis, sickle cells
•Reticulocytosis

51. To establish the diagnosis of sickle cell
anemia
• Sickling test
• Solubility test
• Hb electrophoresis

52. PBS showing Sickle cells

53. Antenatal Diagnosis
• Carried out if the fetus is at risk of thalassemia major or sickle
cell anemia
• DNA analysis by RFLP
• DNA obtained from chorinic villous sampling or from amniotic
fluid

54. Red cell enzyme deficiency
•Abnormalities in the enzymes of the HMP shunt or
glutathione metabolism reduces the ability of red
cells to protect themselves against oxidative injury
•The most important of these is G6PD deficiency
others being pyruvate kinase, glutathione
reductase, glutathione peroxidase

55. G6PD deficiency
Individuals with G6PD deficiency remain asymptomatic under normal
conditions
 Develop life threatening hemolysis when exposed to oxidative drugs,
infection or metabolic derangements.
Hemolysis occurs in 2-3 days of exposure to the oxidant and stops in
a week’s time
Patient presents with sudden pallor, jaundice, with or without fever
and history of drug intake

56. G6PD deficiency
The common clinical syndromes encountered are
• Acute hemolytic anemia
•Neonatal hyperbilirubinemia
•Congenital non spherocytic hemolytic anemia
•Favism

57. Lab diagnosis of G6PD deficiency
• Sudden drop in Hb conc, hematocrit & RBC count
• PBS shows polychromatophils, macrocytes, bite cells and Heinz bodies
• There is reticulocytosis
• Screening tests for enzyme deficiency
Methemoglobin reduction test
 Specific enzyme assay

58. Heinz bodies

59. Immune hemolytic anemia
•Warm antibody type
•Cold antibody type
•Paroxysmal cold hemoglobinuria

60. Warm antibody hemolytic anemia
•Antibody is of Ig G type
•Antibody does not usually fix complement and is
active at 37o C.
• It can be:
Primary(idiopathic)
Secondary :
•Lymphomas, leukemias
•Autoimmune disorders like SLE
•Drugs .

61. Cold antibody type Hemolytic Anemia
•The antibodies are Ig M type
•Antibodies most active in vitro at 00 C to 40 C.
• Antibodies dissociate at 300 C or above
• Agglutination of cells by Ig M and complement fixation
occurs only in peripheral cool parts of the body(fingers,
ears and toes).
•It can be:
Acute : Mycoplasma infection, infectious
mononucleosis
Chronic : Idiopathic or associated with lymphoma

62. Paroxysmal cold hemoglobinuria
•Antibodies are of IgG type
•Antibodies bind to red cells at low
temperature, fix complement causing
hemolysis when the temperature is raised
above 300 C.

63. Autoimmune Hemolytic Anemia

64. When to suspect Autoimmune hemolytic
anemia
•PBS: Spherocytosis , polychromasia & mild
erythroblastemia
•There is reticulocytosis
•Direct antiglobulin test is positive
•This test is utilised to demonstrate antibodies
against RBC by adding antiglobulin ( Coombs)
serum, which combines with antibodies on the
surface of RBC

66. Indirect Coomb’s test

67. SUMMARY

68. PERIPHERAL BLOOD SMEAR EXAMINATION
Spherocytes Microcytes,
hypochromia,
nucleated red cells,
Target cells
Sickle cell, target
cells
Normocytic
normochromic
anemia
Thal syndrome
G6PD, PK
deficiency
screening
positive negative
G6PD/ PK
deficiency
Confirm by enzyme
assay
Coomb’s test
+
HS
AIHA
Osmotic fragility test
+
-
-
Test for
unstable Hb
Sickle cell anemia

69. PERIPHERAL BLOOD SMEAR
Schistocytes
present absent
Valvular
prosthesis
MAHA associated
with defective
valve
Coombs test
positive negative
AIHA Flowcytometry for
PNH
+
PNH
Ovalocytes
H Ovalocytosis
-
D-L test
PCH

70. Thank
You