In this presentation I've tried to summarize classification of hemolytic anemia and in depth review of rbc membrane disorders like hereditary spherocytosis, hereditary elliptocytosis, enzymopathies of hemolytic anemia like g6pd disorder, pyruvate kinase disorders, hemoglobinopathies related to hemolytic anemia like thalassemia, sickle cell anemia and especially pathophysiology and mechanism of hemolysis either extravascular or intravascular. Hope it helps you understand the entity better.

1. Hemolytic Anemia
Dr. Tushar
Santokba Durlabhji Memorial Hospital

2.  ANEMIA - Anemia is defined as reduction of the total
circulating red cell mass below normal limits.
 Anemia reduces the oxygen carrying capacity of
blood, leading to tissue hypoxia.
 Anemia is usually diagnosed based on reduction in
hematocrit and Hemoglobin concentration of the blood to
levels that are below the normal range
 Hemolytic Anemia – Increased red cell destruction (Life
span of RBC could be as low as 15 days)

3.  Red cell destruction occur by 2 mechanisms-
 Extravascular Hemolysis – The site of
destruction is mainly spleen and this is the major
mechanism of red cell hemolysis. Red cells are taken
up by the cells of RE system where they are
destroyed and digested
 Intravascular Hemolysis– This is the minor
pathway of red cell destruction and red cells are
destroyed in circulation releasing hemoglobin.

4. Extravascular Hemolysis
Senescent red cells
Phagocytosed by RE
cells of Spleen
Hemoglobin released
in RE cells and broken
down
Heme + Globin

5. Extravascular Hemolysis
Senescent red cells
Phagocytosed by RE
cells of Spleen
Hemoglobin released
in RE cells and broken
down
Heme + Globin
Heme breaks down into
iron + protoporphyrin
Globin broken down to
amino acids

6. Extravascular Hemolysis
Senescent red cells
Phagocytosed by RE
cells of Spleen
Hemoglobin released
in RE cells and broken
down
Heme + Globin
Heme breaks down into
iron + protoporphyrin
Globin broken down to
amino acids
Re-utilised for
synthesis of α, β chains

7. Extravascular Hemolysis
Senescent red cells
Phagocytosed by RE
cells of Spleen
Hemoglobin released
in RE cells and broken
down
Heme + Globin
Heme breaks down into
iron + protoporphyrin
Globin broken down to
amino acids
Re-utilised for
synthesis of α, β chains
Iron in plasma carried
as transferrin
Biliverdin

8. Extravascular Hemolysis
Senescent red cells
Phagocytosed by RE
cells of Spleen
Hemoglobin released
in RE cells and broken
down
Heme + Globin
Heme breaks down into
iron + protoporphyrin
Globin broken down to
amino acids
Re-utilised for
synthesis of α, β chains
Iron in plasma carried
as transferrin
Biliverdin
Carried to iron storage
sites like bone marrow
Iron stores

9. Extravascular Hemolysis
Senescent red cells
Phagocytosed by RE
cells of Spleen
Hemoglobin released
in RE cells and broken
down
Heme + Globin
Heme breaks down into
iron + protoporphyrin
Globin broken down to
amino acids
Re-utilised for
synthesis of α, β chains
Iron in plasma carried
as transferrin
Biliverdin
Carried to iron storage
sites like bone marrow
Iron stores
Bilirubin
(unconjegated)
Conjugated in liver by
UGT1A1
Bilirubin acted upon by
bacterial enzymes in
instestine
Urobilinogen/Stercobilinogen

10. Intravascular Hemolysis
Red cells in
circulation
Red cells lyse in
circulation
Hemoglobin in
Plasma

11. Intravascular Hemolysis
Red cells in
circulation
Red cells lyse in
circulation
Hemoglobin in
Plasma
Hemoglobinemia
Hb in urine
Combines with
Haptoglobin

12. Intravascular Hemolysis
Red cells in
circulation
Red cells lyse in
circulation
Hemoglobin in
Plasma
Hemoglobinemia
Hb in urine
Combines with
Haptoglobin
Hb absorbed by
kidney tubular cells
Hb converted to
hemosiderin in
tubular cells in few
days
Tubular cells shed
off
Hemosiderinuria
Detected in Urinary deposit by Perl’s
stain

13. Intravascular Hemolysis
Red cells in
circulation
Red cells lyse in
circulation
Hemoglobin in
Plasma
Hemoglobinemia
Hb in urine
Combines with
Haptoglobin
Hb absorbed by
kidney tubular cells
Hb converted to
hemosiderin in
tubular cells in few
days
Tubular cells shed
off
Hemosiderinuria
Detected in Urinary deposit by Pearl’s
stain
Hemoglobinuria
Positive Benzidine
tese

14. Extravascular Hemolysis Intravascular Hemolysis
Site of hemolysis R.E. Cell organs In Circulating blood
S. Methemalbumin -ve +ve
Plasma Hemoglobin -ve +ve
S. Ferritin Normal or ↑ ↓
S. Bilirubin (UC) ↑↑ ↑
S. LDH ↑ ↑↑
Urine Hemoglobin -ve +ve
Urine Hemosiderin -ve +ve
Disease states Thalassemia, Sickle cell
anemia, hereditary
spherocytosis
PNH, G-6-PD deficiency,
black water fever
Tissue iron in spleen,
liver
↑ ↓ or normal
Retic count ↑ ↑
S. Haptoglobin Normal ↓

15. Hereditory Hemolytic Anemia Acquired Hemolytic Anemia
1. Defects in red cell membranes 1. Immunohyemolytic anemia
• Hereditory Sphrecytosis • Autoimmune hemolytic anemia
• Hereditary elliptocytosis  Due to warm antibodies
• Hereditary pyropoikilocytosis. o Idiopathic
• Stamatocytosis o Secondary
• Abetalipoprotenemia  Due to cold antibodies
2. Defects in globin synsthesis o Cold agglutinin disease
• Thalassemias o Paroxysmal cold hemoglobinuria
• Sickling syndromes 2. Fragmentation Syndromes
• Unstable hemoglobins • Hemolytic uremic syndrome
• HbM • Thrombotic thrombocytopenic purpura
• HbD disease • Disseminated intravascular coagulation
• HbE disease • Cardiac hemolytic anemia
• HbQ India 3. Paroxysmal nocturnal hemoglobinuria
• Hemoglobin Lepore 4. Drugs and chemicals
• HbC disase • Oxidant drugs, primaquine. Dapsone
3. Enzyme deficiency of glycolytic pathway 5. Thermal Injury
• Pyruvate kinase deficiency 6. Infections
• Hexokinase deficiency • Clostridium perfringens, Welchiii
4. Enzyme deficiency of pentose phosphate pathway • Bartonellosis
• G-6-P-D deficiency • Cholera
5. Enzyme deficiency of red cell nucleotide metabolism 7. Others

16.  Common findings in Hemolytic anemia
 Pallor, Jaundice, splenomegaly, gall stones skeletal
abnormalities, leg ulcers, dyspnea on exertion,
tachycardia, systolic murmur
 Peripheral Blood Finding
 Polychromatophilia, nucleated red cells,
thrombocytosis and neutrophilia with shift to left.
 Reticulocytosis
 Erythroid hyperplasia in bone marrow

17. Red cell membrane
disorder

18. Overview
Abnormality of
protein
Disorder Comment
Ankyrin HS Common cause of
HS
β Spectrin HS, HE, HPP M/C cause of HS
Protein 4.2 HS Found in Japanese
α Spectrin HS, HE, HPP Spectrin mutation –
common cause of
HE
Band 3 HS Mushroom cells in
PBF
Protein 4.1 HE Found in Arab and
European
population

19. Hereditary Spherocytosis
 Autosomal Dominant (75% cases)
 Autosomal Recessive (ANK-1 & SPTB mutation)
 Spectrin deficiency is the most common (Lower the spectrin content, more
severe is the hemolytic disease clinically and greater number of
spherocytes in blood)
 Spectrin deficiency is associated with reduced membrane stability. These
RBC’s are exposed to shearing stresses, fagments of RBC membrane are
lost, thereby reducing cell surface to volume ratio.
 RBC assume the smallest possible diameter for the volume and become
spherocytes.
 Protein 4.2 (AS) and band 3 deficiency (AD) also result in spectrin
deficiency.

20. normal
Membrane Protrusion
spherocyte
Phagocytosis of microspherocyte
Loss of
membrane
fragment

21. Mechanism of hemolysis in hereditary
spherocytosisSpherocytes
 Reduced deformability
 Greater internal viscosity
 Reduced membrane
plasticity
Difficulty in passing through the inter-
endothelial fenestrations of the venous
sinusoids of spleen

22. Mechanism of hemolysis in hereditary
spherocytosisSpherocytes
Difficulty in passing through the inter-
endothelial fenestrations of the venous
sinusoids of spleen
Lactic acid
accumulates around
the cells
Red cells become conditioned,
spherical and more fragile

23. Mechanism of hemolysis in hereditary
spherocytosisSpherocytes
Difficulty in passing through the inter-
endothelial fenestrations of the venous
sinusoids of spleen
Lactic acid
accumulates around
the cells
Red cells become conditioned,
spherical and more fragile
Phagocytosed by the RE cells of
spleen
Splenomegaly
Increased extravascular hemolysis Anemia,
Reticulocytosis

24. Mechanism of hemolysis in hereditary
spherocytosisSpherocytes
Difficulty in passing through the inter-
endothelial fenestrations of the venous
sinusoids of spleen
Lactic acid
accumulates around
the cells
Red cells become conditioned,
spherical and more fragile
Phagocytosed by the RE cells of
spleen
Splenomegaly
Increased extravascular hemolysis Anemia,
Reticulocytosis
Inhibition of
glycolysis and ↓
ATP generation
Failure of sodium
pump
Accumulation of
water
Abnormal RBC
Phagocytosed by RE cells lining the
sinusoids

25.  Clinical Features
 May present in neonatal period, childhood or adulthood.
 Anemia
 Intermittent Jaundice
 Splenomegaly
 Gall stone
 Chronic leg ulcers
 Classification of severity of Hereditary Spherocytosis (HS)
Hb g/dl
Reticulocyte
Count %
Bilirubin
mg/dl
Spherocytes
on pbf
Spectrin
content % of
Normal
Osmotic
Fragility
Mild HS 11-15 3-10 1-2 Few 80-100 Normal
Moderate 8-11 7-10 1.5-2.5 + 50-80 ↑
Moderately
Severe
6-8 >10 2-3 ++ 40-70 ↑
Severe <6 >12 >3 +++ 20-50 ↑↑

26.  Investigations
 CBC with PBF
 MCHC >36 gm/dl in neonates useful indicator
 PBF – Microspherocytes, Polychromatophilia, reticulocytosis.
 Bone marrow
 Erythroid hyperplasia with a normoblastic marrow
 Biochemical
 S. Bilirubin - ↑
 S. LDH - ↑
 Flow cytometry based EMA binding test (Sensitive and specific)
 Mean fluorescence intensity of EMA tagged RBC is lower in HS (MCF < 0.80)
 Osmotic Fragility test
 Shift to the curve to the right (about 25% of patients may have normal OF)
 Specificity is far lower as compared to EMA
 Incubated Fragility test
 SDS-PAGE analysis of red cell membrane
 Glycerol lysis test
 Osmotic Gradient Ektacytromtery

27. Approach to a case of spherocytosis in peripheral
blood filmSpherocytes on
PBF
Reticulocyte
Counts
High Retic Count
Incubated
osmotic fragility
test
Bite cells in PBF
EMA dye binding
test, SDS-PAGE
for membrane
proteins
Unstable Hb
disease
Coombs test
Immune
mediated
hemolytic
anemia
HS, AIHA
Family History
HS
confirmed
EMA +ve
Heinz bodies +ve
on Retic preparation
G-6-PD assay for
G-6-PD deficiency
Unstable Hb
Isopropanol test
heat denaturation
test+ve
+ve -ve
+ve -ve
Coombs test
+ve
Positive
HS –ve,
AIHA
EM
A-
ve

28.  AD
 β- spectrin, α- spectrin structural defect or deficiency of protein 4.1lead to
changes in membrane function, elliptical shape and mild hemolysis.
 Such cells should be >25% of the red cell population
 Clinically
 Asymptomatic
 Mild hemolytic anemia
 Intermittent jaundice
 Three subtypes
 Typical HE
 Spherocytic HE
 South East Asian Ovalocytosis.
 AD, Seen in Malasia and Phillipines.
 Oval shaped red cells with one or two transverse ridges or
longitudinal slit
 Offer resistant to malaria
Hereditary Elliptocytosis

29.  Rare
 AR
 Defective spectrin gene transmitted by one parent and also an elusive
thalassemia like defect of spectrin synthesis inherited form the normal parent.
 PBF findings
 Poikilocytes
 Fragmented RBC
 Spheroidal RBC
 Elliptocytic RBC
 MCHC is normal
 Incubated osmotic fragility
is increased.
Hereditary Pyropoikilocytosis

30.  Stoma = Fish mouth
 Red cells with a central slit like pallor, Uniconcave/bowl shaped in wet smear
 >30% of red cells (normal individual <5%)
 Two subtype
1) Hereditary stomatocytosis (HSt)
• AD
• Deficiency of membrane protein stomatin located in the band 7 region
result in increased permeability to both Na+ and K+  increased water
content.
• Also called OVERHYDRATED stomatocytosis.
• Associated with RHAG gene mutation.
• C/F – Hemolytic anemia (mild to moderate)
• Reticulocytosis
• MCV raised (110-150 fl)
• Reduced MCHC
• Osmotic Fagility incrased
Stomatocytosis

31. 2) Xerocytosis
• AD
• Increased rate of leakage of K+ ion results in depletion of cations and water
from the red cells which become dehydrated.
• Also called as DEHYDRATED hereditary stomatocytosis. (DHSt)
• C/F – Hemolytic anemia (mild to moderate)
• Target cells, Pyknocytes and echinocytes.
• Jaundice, mild hepatosplenomegaly.

32.  Red cells with 3-12 thorny projection on the cell surface due to
alteration of the lipid composition and fluidity of the red cell membrane.
 Lack central pallor
 Casues
 Abetalipoproteinemia
 Spur cell hemolytic anemia
 HARP synrome
 Post splenectomy
 Neuroacanthocytosis
 Hypothyroidism
 McLeod phenotype of neonatal hepatitis.
Acanthocytosis

33. ENZYMOPATHIES

34.  First enzyme in the hexose monophosphate pathway which protect red cells
from any oxidant injury.
 Sex-linked recessive disease (long arm – band Xq28)
 Highest prevalence in Jews (60-70%) and lowest in Japanese (0.1%), India
(2.6-3%)
 G-6-PD deficiency provide partial protection against Malaria.
 Variants of G-6-PD (WHO classification)
Glucose-6-phosphate dehydrogenase
Deficiency
Class Severity Enzyme Activity Hemolysis
Class I Severe
deficiency
<10% of normal Chronic hemolytic
Anemia
Class II Severe
deficiency
<10% of normal Intermittent
hemolysis
Class III Moderate
deficiency
10-60% of normal On exposure to
drugs
Class IV No deficiency 60-100% of normal No hemolysis
Class V - Increased (>twice
normal)
No hemolysis

35.  G-6-PD B
 Normal G-6-PD enzyme with half life of 62 days
 G-6-PD A
 Deficient enzyme with half life of 13 days
 Enzyme stability is affected
 Seen in Africans
 G-6-PD Mediterranean
 Half life of enzyme is less than 1 day; enzyme is deficient in RBC’s of all
ages.
 Severe hemolysis
 Indian Scenario
 4 common variants
 G6PD mediterranean
 G6PD Kerala-Kalyan
 G6PD Orissa
 G6PD Chatram
 Drug induced hemolysis is seen in patients with G6PD Kerala-Kalyan and

36. Pathophysiology of hemolysis in G-6-PD deficiency
Oxidant stress
Red ells deficient in G-6-PD
Depletion of NADPH and GSH
Oxidant stress in not hydrolysed
because of lack of GSH
Denatured globin, sulfhemoglobin is
formed
Sulf Hb attaches to red cell membrane
forming Heinz bodies
Removal of Heinz bodies in spleen
Extravascular hemolysis
Hemolytic anemia
Lysis of red cells in circulation
Intravascular hemolysis
Hemoglobinemia and
Hemoglobinuria
Hb absorbed by renal tubular cells
Hb hemosiderin inside tubular cells
Hemosiderinuria

37.  Clinical presentation
 Acute Hemolytic Anemia [self limiting], neonatal hyperbilirubinemia
 Pallor, passage of dark urine
 Abdominal pain, fever, chills, jaundice and severe backache
 Favism, Acute renal failure
 Multi organ failure
 Diagnosis
 Hematologic findings
 Anemia with reticulocytosis
 PBF - anisopoikilocytosis with polychromatophilia
Microspherocytes, bite cells, Heinz body, blister cells.
 Urine – Hemoglobinuria, increased urobilinogen
 Methemoglobin reduction test (MRT)
 Ascorbate cyanide test
 Fluorescent spot test
 Cytochemical test
 Dye decolorization test
 Quantitative G-6-PD assay & DNA analysis by PCR

39.  This is the second common enzyme deficiency involving the glycolytic pathway
of red cell metabolism.
 Autosomal recessive condition
 Pyruvate kinase has 2 isoenzymes- PK-L (Liver) and PK-M (Muscles).
 There is accumulation of G-3-P and 2,3-DPG and glucose
 Clinical findings
 Neonatal hyperbilirubinemia
 Pallor, Gall stone, leg ulcers
 Hematological findings-
 moderate anemia with reticulocytosis.
 Peripheral smear demostrates-
 Presence of prickle cells
(red cells having sharp thorn like projections)
 Quantitative assay and DNA analysis by PCR
Pyruvate Kinase Deficiency

40.  Clinically – Mild splenomegaly with intermittent jaundice
 Lab investigation –
 Anemia with reticulocytosis
 Marked Basophilic Stippling
 Bone marrow – Erythroid Hyperplasia with late normoblasts
 Raised S. Bilirubin
Pyrimidine 5’ – Nucleotidase deficiency

41. HEMOGLOBINOPATHI
ES

42. THALASSEMIA
CLASSIFICATION
β thalassemia α thalassemia Misc thalassemia
syndrome
T. Major Hydropes fetais HbS- thalassemia
T. Intermedia Hbh disase HbE- thalassemia
T. Trait A-thalassemia trait HbD-thalassemia
T. Minima α-β-thalassemia
HPHF
γ-Thal, δ-thal

43. Hematological profiles of β Thalassemia
Types of
thalassemia
Hb% MCV MCH
T. Major <7 gm/dl 50-70 fl 12-20 pcg/cell
T. Intermedia 7-10 gm/dl 60-80 fl 15-25 pcg/cell
T. Minor 9-11 gm/dl 65-70 fl 25-30 pcg/cell

44. Pathophysiology of Thalassemia Major

45.  Clinical presentation
 Anemia,
 Progressive increase in pallor
 Protuberant abdomen due to progressive hepatosplenomegaly
 Frontal bossing due to thickening of cranial bone and overgrowth of
zygomatic bones
 Mild jaundice
 Cholelithiasis
 Leg ulcer
 Lab tests
 PBF - Marked anisopoikilocytosis, MCHC
 Tear drop cell and nRBC
 Target cells

47.  Bone marrow
 Hypercellular and demonstrates marked erythroid hyperplasia
 Reversal of M:E ratio to 1:2 or 1:3
 Normoblastic with mild degree of dyserythropoiesis
 Hemosiderin laden macrophages
 Acid elution test for HbF (Kleihauer test)
 S. LDH – high
 S. Haptoglobins – Low
 Globin chain Synthesis (α:β – 2-30:1, normal 1:1)
 Mutation Studies
 Amplification Refractory Mutation System (ARMS) – PCR for point
mutations
 PCR & multiplex PCR
 Minisequencing
 Reverse Dot Blot analysis

48. SICKLING SYNDROME
 Sickle mutation is caused by substitution of valine in place of
glutamic acid in the 6th position (β6 glu-val) ofβ-chain
 Mutation results in clinical presentation
 Sickle cell anemia- HbS-HbS, Homozygous state
 Sickle cell trait -HbA-HbS, heterozygous state
 Sickle cell disease- Refer to all diseases with HbS in
combination with – normal (HbA), abnormal gene of b-
thalassemia, a-thalassemia, HbD, HbE, HbC, HbQ
 Polymerization of deoxygenated HbS is the primary event in the
 pathogenesis of the disease

49. Red cell containing
HbS
Passage through microcirculation of spleen
Low O2 tension
Deoxygenation of HbS and Sickling
Cells pass through circulation with good
O2 tension (Other organs)  Desickling
Various cycles of sickling and desickling
Cell membrane affected and change in
membrane permeability
Irreversible sickling
Sickled RBCs Macrophage PhagocytosisIncreased mechanical
fragility
Extravascular hemolysisIntravascular hemolysis

50.  Clinical features
 Delay in puberty, growth and development
 Recurrent leg ulcers
 Avascular necrosis of femur head
 Dactylitis ( Hand –Foot syndrome )
 Pneumonia, meningitis, Osteomylitis
 Jaundice and liver enlargement
 Pigment gall stones
 Acute abdominal pain ( infarcts of abdominal viscera)
 Priapism
 Acute chest syndrome
 Sickle retinopathy- Salmon patches- intra retinal hemmorhages

51.  Lab Investigation
 Anemia- moderately severe anemia with Hb 5-10 gm
 PBF demonstrates –
 Normocytic normochromic to mildly hypochromic moderate to severe
degree of anisopoikliocytosis.
 Sickle cells, target cells, ovalocytes, polychromtophila with nucleted
RBCs. Howell-jolly bodies also seen
 TLC- Mildly elevated ; Platelets- Increased
 Reticulocytosis- 3%-10%

52.  Sickling tests- Presence of HbS demostrated by using reducing agent
like 2% sodium metabisulphite.
 Sickling solubility test
 Hb electrophoresis- HbS is a slow moving Hb as compared to HbA and
HbF. However, electrophoretic mobility of HbD/HbQ india is similar to
HbS, therefore sickling test is essential to differentiate.
 HPLC- On HPLC, HbS has a retention time of 4.40 to 4.50 min, while
HbD punjab is is 4.50-4.15 min.

53. FRGAMENTATION
SYNDROME

54. A. Cardiac Hemolytic Anemia D. Microangipathic Hemolytic
Anemia
• Valvular prosthesis • HUS
• Aortic stenosis • TTP
• Mitral valve disease • Pre-eclampsia
• Coarctation of aorta • HELLP
B. March Hemoglobinuria • Drugs
C. Non-MAHA genetic/acquired
RBC disorders
• Mucin producing
adenocarcinoma
• Thalassemia • Transplantation
• Megaloblastic anemia • Malignant HTN
• Primary myelofibrosis • Vasculitis
• Thermal Injury • Disseminated malignancies
• Associated with DIC

55. Thank You