This document summarizes different types of hemolytic anemias. It describes the features shared by hemolytic anemias such as a shortened red blood cell lifespan and increased erythropoiesis. Two main types of hemolysis are discussed: extravascular hemolysis where red blood cells are destroyed within macrophages, and intravascular hemolysis where they lyse within blood vessels. Specific causes of hemolysis are then outlined, including inherited disorders like hereditary spherocytosis and enzyme deficiencies like glucose-6-phosphate dehydrogenase deficiency. Features of acquired conditions like paroxysmal nocturnal hemoglobinuria and immune-mediated hemolytic anemias are also summarized.

1. Presented by :Dr. Prakash Poudel
2074/03/02

2.  Hemolytic anemias share the following features:
1. A shortened red cell life span below the normal 120
days
2. Elevated erythropoietin levels and a compensatory
increase in erythropoiesis
3. Accumulation of hemoglobin degradation products
that are created as part of the process of red cell
hemolysis

3.  Extravascular hemolysis:
 Premature destruction of red cells occurs within
macrophages of the reticuloendothelial system, e.g. in the
bone marrow, liver and spleen
 C/F: Anemia, splenomegaly, and jaundice.
Causes include RBC membrane abnormalities such as
 Membrane bound immunoglobulin,
 Hereditary spherocytosis
 Hereditary elliptocytosis
 Extravascular hemolysis is characterized by spherocytes.

4. spherocytes.

5. Intravascular hemolysis
 In intravascular hemolysis RBCs lyse in the circulation
releasing hemoglobin into the plasma
Lab features of intravascular hemolysis.
 Raised plasma and urine haemoglobin,
 Positive serum (Schumm) test for methaemalbumin,
 Presence of haemosiderin in urine.
 Decreased haptoglobin level.
 C/F: Anemia, hemoglobinemia, hemoglobinuria,
hemosiderinuria, and jaundice

6. .Causes of intravascular haemolysis
 mechanical injury, (March haemoglobinuria)
 complement fixation (PNH)
 intracellular parasites (e.g., falciparum malaria) or
exogenous toxic factors.
• Mismatched blood transfusion (usually ABO)
• G6PD deficiency with oxidant stress
• Red cell fragmentation syndromes

7. schistocytes. Urine sample (Perl stain). Hemosiderin
deposition (blue) in sloughed epithelial cells
of renal tubules occurs in patients who have
had intravascular hemolysis

8. .
Peripheral smear schistocytes spherocytes
Haptoglobin decrease/absent mild decrease
Urine hemosiderin ++ negative
Urine hemoglobin ++ negative
Direct DAT usually negative ++++
LDH increase increase
Intravascular hemolysis Extravascular hemolysis

10. Hemolytic anemias in general
 Peripheral blood
 In common polychromasia and an increased reticulocyte
count.
 Macrocytosis in chronic and severe hemolytic anemia
 Other features depend on nature of anemia
 Bone marrow cytology
 Hypercellular due to erythroid hyperplasia.
 In some patients, fat cells are totally lost.
 Haemopoiesis is often macronormoblastic(Large normoblast).
 Some cases have quite marked dyserythropoiesis.
 In Extravascular hemolysis iron stores are commonly increased
but deplete in intravascular hemolysis.

11. BM aspirate film,autoimmune haemolytic anaemia,
showing an erythroid island composed of erythroblasts
clustered around a debris - laden macrophage.

12. Classification.
A. Inherited genetic defects
 Red cell membrane disorders
 Hereditary spherocytosis,
 Hereditary elliptocytosis
 Enzyme deficiencies
 Hexose monophosphate shunt enzyme deficiencies: G6PD
deficiency, glutathione synthetase deficiency
 Glycolytic enzyme deficiencies :Pyruvate kinase deficiency,
hexokinase deficiency
 Hemoglobin abnormalities
 Deficient globin synthesis :-Thalassemia syndromes
 Structurally abnormal globins (hemoglobinopathies) Sickle
cell disease, unstable hemoglobins

13. .
B. Acquired genetic defects
 Deficiency of phosphatidylinositol-linked glycoproteins
 Paroxysmal nocturnal hemoglobinuria
 Antibody-mediated destruction
 Hemolytic disease of the newborn (Rh disease), transfusion
reactions, drug-induced, autoimmune disorders
C. Mechanical trauma
 Microangiopathic hemolytic anemias
 Hemolytic uremic syndrome, (HUS)
 Disseminated intravascular coagulation, (DIC)
 Thrombotic thrombocytopenianpurpura (TTP)
 Cardiac traumatic hemolysis
 Defective cardiac valves
 Repetitive physical trauma
 Bongo drumming, marathon running, karate chopping

14. D.Infections of red cells
 Malaria, babesiosis
E. Toxic or chemical injury
 Clostridial sepsis, snake venom, lead poisoning
F. Membrane lipid abnormalities
 Abetalipoproteinemia, severe hepatocellular liver
disease
G. Sequestration
 Hypersplenism

15. A. Inherited genetic defects
 Hereditary Spherocytosis
 It is an inherited disorder caused by intrinsic defects in
RBC membrane skeleton that gives spheroid shape,
less deformable, and vulnerable to splenic
sequestration and destruction.
 Ankyrin, band 3, spectrin, or band 4.2 proteins are
essential to maintain membrane integrity.
 Mutation in these proteins cause red cells to lose
membrane fragments ---- ratio of surface area to
volume decrease --- cells adopt a spherical shape.
 Spherocytic cells are less deformable therefore become
trapped in the splenic cords---phagocytosed

17. PBS
Spherocytosis: small, dark-staining (hyperchromic)
red cells lacking the central zone of pallor
 Other features: reticulocytosis,
 Bone Marrow : erythroid hyperplasia,
 Hemosiderosis, and mild jaundice.
 Cholelithiasis (pigment stones)
 Moderate splenomegaly
 Spherocytosis also seen autoimmune hemolytic
anemia

19. Other lab investigations
Osmotic fragility test:osmotic lysis when incubated in
hypotonic salt solutions, which causes the influx of
water into spherocytes with little margin for
expansion.
 Increased MCHC due to dehydration caused by the loss
of K+ and H2O.
 Flow cytometry: Eosine-5 maleimide (EMA) test for
Band 3 abnormality.

20. Enzyme abnormalities
 Glucose-6-Phosphate Dehydrogenase Deficiency
 Main function of G6PD is to protect the red cell against oxidative injury

21. .
 The episodic hemolysis is due to exposure to oxidant
stress. Eg: Infections, antimalerial drugs,fava beans.
 Oxidants cause both intravascular and extravascular
hemolysis in G6PD-deficient individuals.
 Oxidants causes the cross-linking of reactive sulfhydryl
groups on globin chains, which become denatured to
form Heinz bodies
 Heinz bodies can damage the membrane sufficiently to
cause intravascular hemolysis.
 As red cells pass through the splenic cords,
macrophages pluck out the Heinz bodies–-membrane
damage-- Bite cell.
 Less severely damaged cells become spherocytes
 Both bite cells and spherocytes are removed by spleen.

22.  Intravascular hemolysis characterised by anemia,
hemoglobinemia, and hemoglobinuria
 Only older red cells are at risk for lysis so the episode is
self-limited
 Hemolysis ceases when only younger G6PD-replete
red cells remain
 PBS:‘bite’ and ‘blister’ cells and polychromasia
 Heinz bodies (denatured haemoglobin) may be seen
in a reticulocyte preparation with supravital staining.

24. Hemoglobin abnormalities
Sickle Cell Disease
 Sickle cell disease is a common hereditary
hemoglobinopathy caused by a point mutation in β-globin
that promotes the polymerization of deoxygenated
hemoglobin, leading to red cell distortion, hemolytic
anemia, microvascular obstruction, and ischemic tissue
damage.
 Point mutation in the sixth codon of β-globin that leads to
the replacement of a glutamate by valine.
 Pathogenesis. The major pathologic manifestations—
chronic hemolysis, microvascular occlusions, and tissue
damage—due to stacking of HbS into polymers when
deoxygenated.

26. LABORATORY
FINDINGS/DIAGNOSIS
 Hb% : 6-10 mg/dl
 MCV : NORMAL
 MCHC: NORMAL
 PBS:
 Red cells: normocytic normochromic, variable
numbers of sickled forms, target cells, cigar-shaped
cells, and ovalocytes .
 The morphologic features of accelerated
erythropoiesis, which include polychromatophil,
basophilic stippling, and nRBCs, are prominent.
 Howell-Jolly bodies and Pappenheimer bodies
(reflect functional asplenia)

27. .
 WBC count: increase
 Platelet count: increase
 Bone marrows(usually not done): erythroid hyperplasia
 ESR: consistently low (because of the failure of sickle
cells to undergo rouleaux formation.)
 The level of serum alkaline phosphatase increases
during symptomatic crises
 Sickling test : Positive
 HbS Solubility test : positive
 Other diagnostic test:
 Electrophoresis
 HPLC
Genotype Mean
Hb%
MCV S A F A2 other
SS 8.1 N 75-95 -------- 1-20 2-5 --------

29. Thalassemia
 Autosomal recessive syndromes divide into α- and β-
thalassaemia depending on whether there is reduced
synthesis of α- or β-globin
 α-Thalassaemia
 Normally there are four α-globin genes, two on each
chromosome 16 . The severity of α-thalassaemia depends
on the number of α-genes deleted or, less frequently,
dysfunctional.
 The β-thalassemias: are caused by mutations that
diminish the synthesis of β-globin chain
 Splicing mutations.
 Promoter region mutations.
 Chain terminator mutations.

30. Point mutation
Gene deletion

31. .
 PBS:
 Striking hypochromia, microcytosis, anisocytosis and
poikilocytosis. Basophilic stippling, Pappenheimer bodies and
dysplastic circulating erythroblasts are also present.
 If the patient has been transfused, the blood film is
dimorphic.several nRBCs.
 Bonemarrow
 Marked erythroid hyperplasia, severe erythroid dysplasia and
poor haemoglobinization.
 Some erythroblasts contain cytoplasmic inclusions.
 Marrow expansion erodes cortical bone an induces new bone
formation, giving rise to “crewcut” appearance on x-ray.
 Spleen enlargement due to phagocyte hyperplasia and
extramedullary hematopoiesis leads to Liver and LN
enlargement
 Hemosiderosis and secondary hemochromatosis in heart,
liver, and pancreas.

32. BM aspirate film, β thalassaemia major, showing
erythroid hyperplasia and dyserythropoiesis (bilobed
and irregular nuclear membrane). Several cells
contain cytoplasmic inclusions, composed of
precipitated ฀α chains.

33. β-Thalassemia major.: nRBCs, microcytosis, and hypochromasia target cells,
teardrop cells, fragments, basophilic stippling, and Pappenheimer bodies.

34. HbH inclusions. Peripheral blood stained
with supravital stain brilliant cresyl blue.

35. . Acquired genetic defects
Paroxysmal Nocturnal
Hemoglobinuria (PNH)
 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 membrane-associated complement regulatory
proteins.
 GPI is anchor which links signaling proteins (protective
proteins-CD55,CD59 )to the cell membrane.
 These protein has function of complement degradation.
 Red cells deficient in these GPI-linked factors are
abnormally susceptible to lysis or injury by complement.

36.  C/F
 Paroxysmal and nocturnal hemolysis(25%)
 Chronic hemolysis –more common
 Decrease in blood pH during sleep increases the
activity of complement leads to hemolysis.
 Anemia: mild to moderate .
 Hemosiderinuria eventually leads to iron deficiency,
which can exacerbate the anemia if untreated.
 Thrombosis of hepatic, portal,cerebral vein is the
leading cause of death

37.  Diagnosed by flow cytometry by detecting red cells
that are deficient in GPI-linked proteins such as CD59
and CD55.
Normal individual PNH

38. Antibody-mediated destruction/
Immunohemolytic Anemias
 Caused by antibodies that bind to red cells, leading to their
premature destruction.

39. Warm Antibody Type (IgG active at 37C)
 Most common form of immunohemolytic anemia.
 Most causative antibody IgG and IgA (less
commonly)
 IgG-coated red cells bind to Fc receptors on
phagocytes, which remove red cell membrane
during “partial” phagocytosis.
 This partial loss of membrane leads to formation
of spherocytes.

40. .
Mechanisms of drug-induced immunohemolytic
anemia
1.Antigenic drugs(penicillin,cephalosporins)
 Bind to the red cell membrane and are recognized by
antidrug antibodies.
 Antibodies recognize a complex of the drug and a
membrane protein—fix complement –intravascular
hemolysis ,OR, Opsonization occurs– extravascular
hemolysis in phagocytes
2. Tolerance-breaking drugs(α-methyldopa)
 By unknown mechanism produce antibodies against red cell
antigen (especially against Rh antigen)

41. Cold Agglutinin Type.(IgM type)
 IgM antibodies bind RBC at low temperatures
 Transient Cold antibodies production in: Mycoplasma
pneumoniae, EBV,CMV, influenza virus, HIV.
 Chronic cold antibodies in: B cell neoplasm,idiopathic.
 Below 30°C in exposed fingers, toes, and ears IgM bind to
RBCs for a transient period which deposits C3b(opsonin) on
RBCs---removal of affected red cells by phagocytes in the
spleen, liver, and bone marrow.
 Vascular obstruction caused by agglutinated red cells results
in pallor, cyanosis, and Raynaud phenomenon in body parts
exposed to cold temperature
 Seen in children following viral infections

42. Cold agglutinin.:The red blood cells form clumps in which distinguishing
the borders of individual erythrocytes is difficult.

43. Cold Hemolysin Type (IgG )
 Paroxysmal cold hemoglobinuria
 IgG type autoantibodies that bind to the P blood group
antigen on the red cell surface in cool, peripheral regions of
the body.
 Complement-mediated lysis occurs when the cells
recirculate to warm central regions, because the
complement cascade functions more efficiently at 37°C.

44. Immunehemolytic anemia
 PBS:Blood film shows microspherocytes,
polychromasia, ± circulating nRBCs
 Direct antiglobulin test (DAT) (Coomb’s test) is
positive

45. Coombs test
Direct Coombs antiglobulin test:
Patient’s red cells are mixed with sera containing antibodies
that are specific for human immunoglobulin or complement.
 If either immunoglobulin or complement is present on the
surface of the red cells, the antibodies cause agglutination,
which is easily appreciated visually as clumping.
Indirect Coombs antiglobulin test,
 The patient’s serum is tested for its ability to agglutinate
commercially available red cells bearing particular defined
antigens.
 Red cells are first incubated with patient’s serum at 37°C for
30 minutes . DAT is then performed, which will be positive if
there are antibodies in the serum reacting against the red
blood cells

46. Direct Coombs test

47. Mechanical trauma
 Microangiopathic hemolytic anemias
 Hemolytic uremic syndrome, (HUS)
 Disseminated intravascular coagulation,(DIC)
 Thrombotic thrombocytopenic purpura( TTP)
 Cardiac traumatic hemolysis
 Defective cardiac valves
 Repetitive physical trauma
 Bongo drumming, marathon running, karate chopping

48. .
• Hemolytic uremic syndrome (HUS)
 Occurs acutely and sporadically after GIT infections with by
Escherichia coli strain O157:H7, which elaborates a Shiga-
like toxin.
 Pathophysiology:
 Typical HUS: Endothelial injury by toxins or antibodies and
endothelial activation cause intravascular thrombosis and
platelet aggregation, which causes vascular obstruction and
vasoconstriction, affects predominantly the renal vessels;
 Atypical HUS: There is excessive complement activation in
microvasculature due to alternative complement pathway
inhibitor deficiencies
 Complement factor H, membrane cofactor protein(CD46)
or factor I
 Fibrin / red cell rich thrombi

49. Schistocytes demonstrated in Microangiopathic hemolytic anemia

50. Thrombocytopenic Purpura (TTP)
 Occurs due to enhanced platelet aggregation,
associated with a deficiency of enzyme ADAMTS13
(also called vWF metalloprotease) which normally
degrades very high-molecular-weight multimers of
vWF
 In absence of ADAMTS13 , vWF accumulate in
plasma and tend to promote platelet activation
and aggregation
 Platelet rich thrombi

51. PBS of patient with TTP: RBC fragments and
profound thrombocytopenia.

53. .
Disseminated Intravascular Coagulation
(DIC)
 DIC is an acute, subacute, or chronic thrombohemorrhagic
disorder characterized by the excessive activation of
coagulation and the formation of thrombi in the
microvasculature of the body.
 Two major mechanisms trigger DIC:
(1) release of tissue factor or other, poorly characterized
procoaagulants, into the circulation, and
(2) widespread injury to the endothelial cells.
 Can produce bleeding, vascular occlusion and tissue
hypoxemia, or both
 Common triggers: sepsis, major trauma, certain cancers,
obstetric complications

54. Pathophysiology of DIC

55. Possible consequences of DIC
 Widespread deposition of fibrin within the microcirculation
leads to ischemia of vulnerable organs.
 Microangiopathic hemolytic anemia results from the
fragmentation of red cells as they squeeze through the
narrowed microvasculature.
 Consumption of platelets and clotting factors and the
activation of plasminogen, leading to a hemorrhagic
diathesis.
 Fibrin degradation products inhibit platelet aggregation,
fibrin polymerization, and inhibit thrombin.

56. Cardiac traumatic hemolysis
 Artificial mechanical cardiac valves are more
frequently implicated than are bioprosthetic porcine
or bovine valves.
 The hemolysis stems from shear forces produced by
turbulent blood flow and pressure gradients across
damaged valves.

57. March haematuria
 March hematuria, occurs when blood is seen in the
urine after repetitive impacts on the body, particularly
affecting the feet.
 The word "march" is in reference to the condition
arising in soldiers who have been marching for long
periods.

58.  Thank you