1. Hemolytic Anemia
Dr. Rafi Ahmed Ghori
FCPS
Professor Medicine
Liaquat University of Medical & Health Sciences,
Jamshoro

2. Classification of Hemolytic
Anemias
1. Abnormalities of RBC interior
1. Enzyme defects.
2. Hemoglobinopathies
2. RBC membrane abnormalities
1. Hereditary spherocytosis etc.
2. Paroxysmal nocturnal hemoglobinuria.
3. Spur cell anemia.
3. Extrinsic factor
1. Hypersplenism.
2. Antibody: immune hemolysis.
3. Microangiopathic hemolysis.
4. Infections, toxins, etc.
Intracorpuscular
Extracorpuscular
Hereditary
Acquired
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3. Red Cell Membrane Disorders
Usually detected by morphologic
abnormalities of RBC on blood film.
Three types of inherited RBC membrane
abnormalities
– Hereditary sphercytosis.
– Hereditary elliptocytosis (including hereditary
pyropoikilocytosis).
– Hereditary stomatocytosis.

4. Red cell membrane has a lipid bilayer to which a
‘skeleton’ of filamentous proteins is attached via
special linkage proteins.

5. RED CELL CYTOSKELETON

6. HEREDITARY
SPHEROCYTOSIS
 Defective or absent spectrin molecule
 Leads to loss of RBC membrane, leading to
spherocytosis
 Decreased deformability of cell
 Increased osmotic fragility
 Extravascular hemolysis in spleen

8. SPLENIC ARCHITECTURE

9. HEREDITARY
SPHEROCYTOSIS
Osmotic Fragility
0
20
40
60
80
100
0.3 0.4 0.5 0.6
NaCl (% of normal saline)
%Hemolysis
Normal HS

10. Hereditary Spherocytosis
Usually has an autosomal dominant
inheritance pattern and incidence of
approximately 1:1000 to 1:4500.
In ~20% patients, the absence of
hematologic abnormalities in family
members suggests either autosomal
recessive inheritance or spontaneous
mutation.

11. Hereditary Spherocytosis
Sometimes clinically apparent in early
infancy but often escapes detection until
adult life.

12. Hereditary Spherocytosis
Clinical Manifestations
– Anemia.
– Splenomegaly.
– Jaundice.

13. Hereditary SpherocytosisClinical Manifestations
– Prominent jaundice accounts for disorder’s
prior designation as “congenital hemolytic
jaundice” and is due to an increased
concentration of unconjugated (indirect-
reacting) bilirubin in plasma.

14. Hereditary Spherocytosis
Clinical Manifestations
– Jaundice may be intermittent and tends to be less
pronounced in early childhood.
– Due to increased bile pigment production,
pigmented gallstones are common, even in
childhood.
– Clinical course may be complicated by crisis
Hemolytic crisis (infection).
Megaloblastic crisis (folate deficiency, especially in
pregnancy).
Aplastic crisis (parvovirus infection).

15. Hereditary SpherocytosisDiagnosis
– Must be distinguished primarily from the
spherocytic hemolytic anemias associated with
RBC antibodies.
– If present, family history of anemia and/or
splenectomy.
– Immune spherocytosis  positive direct
Coombs test.

16. Hereditary SpherocytosisDiagnosis
– Splenomegaly can also be seen
Cirrhosis.
Clostridial infections.
G6PD deficiency.

17. Hereditary Spherocytosis
Treatment
– Splenectomy reliably corrects the anemia.
– Splenectomy in children should be postponed
until the age of 4-years.
– Polyvalent pneumococcal vaccine should be
administered at least 2-weeks before
splenectomy.
– In patients with severe hemolysis  folic acid
(5-mg/d) should be administered
prophylactically.

18. Hereditary SpherocytosisTreatment
– Acute, severe hemolytic crises requires
transfusion.

19. Hereditary ElliptocytosisHeterogeneous disorders in elliptocytic red
cells.
Functional abnormality of anchor proteins
in red cell membrane (alpha spectrin or
protein 4.1).
Autosomal dominant or recessive.
Less common than hereditary spherocytosis
(1/10,000).

20. Red cell membrane has a lipid bilayer to which a
‘skeleton’ of filamentous proteins is attached via
special linkage proteins.

21. Hereditary ElliptocytosisClinical presentation depends upon degree
of membrane defect.
Asymptomatic diagnosed on blood film.
Chronic compensated hemolytic state.

22. Hereditary ElliptocytosisTreatment
– Same as hereditary spherocytosis only in
chronic hemolytic anemia.
Differential diagnosis includes
– Iron deficiency anemia
– Thalassaemia.
– Mylofibrosis.
– Mylodysplasia.
– Pyruvate kinase deficiency.

23. Paroxysmal Nocturnal
Hemoglobinuria
 Clonal cell disorder
 Ongoing Intra- & Extravascular hemolysis; classically
at night
 Testing
– Acid hemolysis (Ham test)
– Sucrose hemolysis
– CD-59 negative (Product of PIG-A gene)
 Acquired deficit of GPI-Associated proteins
(including Decay Activating Factor)

25. GPI BRIDGE

26. Paroxysmal Nocturnal
Hemoglobinuria
GPI Proteins
 GPI links a series of proteins to outer leaf of cell
membrane via phosphatidyl inositol bridge, with
membrane anchor via diacylglycerol bridge
 PIG-A gene, on X-chromosome, codes for synthesis
of this bridge; multiple defects known to cause lack
of this bridge
 Absence of decay accelerating factor leads to failure
to inactivate complement & thereby to increased cell
lysis

27. HEMOLYTIC ANEMIA
Membrane abnormalities -
Enzymopathies
 Deficiencies in Hexose Monophosphate
Shunt
– Glucose 6-Phosphate Dehydrogenase Deficiency
 Deficiencies in the EM Pathway
– Pyruvate Kinase Deficiency

28. G6PD DEFICIENCY
Function of G6PD
G6PD
GSSG 2 GSH
NADPH NADP
2 H2O H2O2
6-PG G6P
Hgb
Sulf-Hgb
Heinz bodies
Hemolysis
Infections
Drugs

29. Glucose 6-Phosphate
Dehydrogenase
Functions
 Regenerates NADPH, allowing regeneration of
glutathione
 Protects against oxidative stress
 Lack of G6PD leads to hemolysis during oxidative
stress
– Infection
– Medications
– Fava beans
 Oxidative stress leads to Heinz body formation, 
extravascular hemolysis

30. Glucose 6-Phosphate
Dehydrogenase
Different Isozymes
0
0.2
0.4
0.6
0.8
1
0 20 40 60 80 100 120
RBC Age (Days)
G6PDActivity(%)
Normal (GdB) Black Variant (GdA-)
Mediterranean (Gd Med)
Level needed for protection vs ordinary oxidative stress

32. HEMOLYTIC ANEMIA
Causes
 INTRACORPUSCULAR HEMOLYSIS
– Membrane Abnormalities
– Metabolic Abnormalities
– Hemoglobinopathies
 EXTRACORPUSCULAR HEMOLYSIS
– Nonimmune
– Immune

33. EXTRACORPUSCULAR
HEMOLYSIS
Nonimmune
 Mechanical
 Infectious
 Chemical
 Thermal
 Osmotic

34. Microangiopathic Hemolytic
Anemia
Causes
 Vascular abnormalities
– Thrombotic thrombocytopenic purpura
– Renal lesions
 Malignant hypertension
 Glomerulonephritis
 Preeclampsia
 Transplant rejection
– Vasculitis
 Polyarteritis nodosa
 Rocky mountain spotted fever
 Wegener’s granulomatosis

35. Microangiopathic Hemolytic
Anemia
Causes - #2
– Vascular abnormalities
 AV Fistula
 Cavernous hemangioma
 Intravascular coagulation predominant
– Abruptio placentae
– Disseminated intravascular coagulation

37. IMMUNE HEMOLYTIC ANEMIA
General Principles
 All require antigen-antibody reactions
 Types of reactions dependent on:
– Class of Antibody
– Number & Spacing of antigenic sites on cell
– Availability of complement
– Environmental Temperature
– Functional status of reticuloendothelial system
 Manifestations
– Intravascular hemolysis
– Extravascular hemolysis

38. IMMUNE HEMOLYTIC ANEMIA
General Principles - 2
 Antibodies combine with RBC, & either
1. Activate complement cascade, &/or
2. Opsonize RBC for immune system
 If 1, if all of complement cascade is fixed to red cell,
intravascular cell lysis occurs
 If 2, &/or if complement is only partially fixed,
macrophages recognize Fc receptor of Ig &/or C3b
of complement & phagocytize RBC, causing
extravascular RBC destruction

39. IMMUNE HEMOLYTIC
ANEMIA
Coombs Test - Direct
 Looks for immunoglobulin &/or complement of
surface of red blood cell (normally neither found on
RBC surface)
 Coombs reagent - combination of anti-human
immunoglobulin & anti-human complement
 Mixed with patient’s red cells; if immunoglobulin or
complement are on surface, Coombs reagent will link
cells together and cause agglutination of RBCs

40. IMMUNE HEMOLYTIC
ANEMIA
Coombs Test - Indirect
 Looks for anti-red blood cell antibodies in the
patient’s serum, using a panel of red cells with
known surface antigens
 Combine patient’s serum with cells from a panel
of RBC’s with known antigens
 Add Coombs’ reagent to this mixture
 If anti-RBC antigens are in serum, agglutination
occurs

41. HEMOLYTIC ANEMIA -
IMMUNE
 Drug-Related Hemolysis
 Alloimmune Hemolysis
– Hemolytic Transfusion Reaction
– Hemolytic Disease of the Newborn
 Autoimmune Hemolysis
– Warm autoimmune hemolysis
– Cold autoimmune hemolysis

42. IMMUNE HEMOLYSIS
Drug-Related
 Immune Complex Mechanism
– Quinidine, Quinine, Isoniazid
 “Haptenic” Immune Mechanism
– Penicillins, Cephalosporins
 True Autoimmune Mechanism
– Methyldopa, L-DOPA, Procaineamide, Ibuprofen

43. DRUG-INDUCED
HEMOLYSIS
Immune Complex Mechanism
 Drug & antibody bind in the plasma
 Immune complexes either
– Activate complement in the plasma, or
– Sit on red blood cell
 Antigen-antibody complex recognized by RE system
 Red cells lysed as “innocent bystander” of destruction
of immune complex
 REQUIRES DRUG IN SYSTEM

44. DRUG-INDUCED
HEMOLYSIS
Haptenic Mechanism
 Drug binds to & reacts with red cell surface
proteins
 Antibodies recognize altered protein, ± drug,
as foreign
 Antibodies bind to altered protein & initiate
process leading to hemolysis

45. DRUG-INDUCED
HEMOLYSIS
True Autoantibody Formation
 Certain drugs appear to cause antibodies
that react with antigens normally found on
RBC surface, and do so even in the absence
of the drug

46. ALLOIMUNE HEMOLYSIS
Hemolytic Transfusion Reaction
 Caused by recognition of foreign antigens on transfused
blood cells
 Several types
– Immediate Intravascular Hemolysis (Minutes) - Due to preformed
antibodies; life-threatening
– Slow extravascular hemolysis (Days) - Usually due to repeat
exposure to a foreign antigen to which there was a previous
exposure; usually only mild symptoms
– Delayed sensitization - (Weeks) - Usually due to 1st exposure to
foreign antigen; asymptomatic

47. INCOMPATIBLE RBC
TRANSFUSION
Rate of Hemolysis
0
20
40
60
80
100
0 1 2 3 4 5 6 7
Weeks Post-Transfusion
SurvivingCells(%)
Normal Immediate Intravascular Hemolysis
Slow Extravascular Hemolysis Delayed Extravascular Hemolysis

48. ALLOIMMUNE HEMOLYSIS
Testing Pre-transfusion
 ABO & Rh Type of both donor & recipient
 Antibody Screen of Donor & Recipient,
including indirect Coombs
 Major cross-match by same procedure
(recipient serum & donor red cells)

49. ALLOIMMUNE HEMOLYSIS
Hemolytic Disease of the Newborn
 Due to incompatibility between mother negative for
an antigen & fetus/father positive for that antigen.
Rh incompatibility, ABO incompatibility most
common causes
 Usually occurs with 2nd or later pregnancies
 Requires maternal IgG antibodies vs. RBC antigens
in fetus

50. ALLOIMMUNE HEMOLYSIS
Hemolytic Disease of the Newborn -
#2
 Can cause severe anemia in fetus, with
erythroblastosis and heart failure
 Hyperbilirubinemia can lead to severe brain damage
(kernicterus) if not promptly treated
 HDN due to Rh incompatibility can be almost totally
prevented by administration of anti-Rh D to Rh
negative mothers after each pregnancy

51. AUTOIMMUNE HEMOLYSIS
 Due to formation of autoantibodies that
attack patient’s own RBC’s
 Type characterized by ability of
autoantibodies to fix complement & site of
RBC destruction
 Often associated with either
lymphoproliferative disease or collagen
vascular disease

52. AUTOIMMUNE HEMOLYSIS
Warm Type
 Usually IgG antibodies
 Fix complement only to level of C3,if at all
 Immunoglobulin binding occurs at all temps
 Fc receptors/C3b recognized by macrophages; 
 Hemolysis primarily extravascular
 70% associated with other illnesses
 Responsive to steroids/splenectomy

53. AUTOIMMUNE HEMOLYSIS
Cold Type
 Most commonly IgM mediated
 Antibodies bind best at 30º or lower
 Fix entire complement cascade
 Leads to formation of membrane attack complex,
which leads to RBC lysis in vasculature
 Typically only complement found on cells
 90% associated with other illnesses
 Poorly responsive to steroids, splenectomy;
responsive to plasmapheresis

54. HEMOLYTIC ANEMIA
Summary
 Myriad causes of increased RBC destruction
 Marrow function usually normal
 Often requires extra folic acid to maintain
hematopoiesis
 Anything that turns off the bone marrow can
result in acute, life-threatening anemia