Hemolytic anemia

HEMOLYTIC ANEMIA
Presented by
Dr. PANKAJ YADAV
drpankajyadav05@gmail.com

Normal Red Cells
No nucleus
Biconcave discs
Center 1/3 pallor
Pink cytoplasm (Hb filled)
Cell size 7- 8 µ - capill.
Negative charge
100-120 days life span

The Factory – Bone Marrow
Sternum, pelvis, vertebrae, long
bones, skull bones, Tibia (paed)
From stem cells (pleuripotent)
75% of marrow for WBC
25% of BM for Red cells
Erythrod / Granulocyte Ratio 1:3
Large white areas are marrow
fat

drpankajyadav05@gmai
l.com
Hemoglobin (Hb)

ERYTHROPOIESIS

HEMOPOIESIS
• Hemo: Referring to blood cells
• Poiesis: “The development or production of”
• The word Hemopoiesis refers to the production &
development of all the blood cells:
– Erythrocytes: Erythropoiesis
– Leucocytes: Leucopoiesis
– Thrombocytes: Thrombopoiesis.
• Begins in the 20th week of life in the fetal liver & spleen,
continues in the bone marrow till young adulthood &
beyond!

Sites of erythropoiesis
• Mesoblastic stage-
in the yolk sac
Starts at 2 weeks of intrauterine life
• Hepatic stage-
2-7 months
Both liver and spleen
• Myeloid stage

Myeloid stage
• Occurs in bone marrow
• Starts at 5 months of fetal life and takes over
completely at birth
• Red bone marrow of all bone.
• Late adult life, red marrow of flat bones

SITES OF HEMOPOIESIS
• Active Hemopoietic
marrow is found, in
children throughout the:
– Axial skeleton:
• Cranium
• Ribs.
• Sternum
• Vertebrae
• Pelvis
– Appendicular skeleton:
• Bones of the Upper &
Lower limbs
• In Adults active
hemopoietic marrow is
found only in:
– The axial skeleton
– The proximal ends of the
appendicular skeleton.

In adults extramedullary hematopoiesis may occur in
diseases in which the bone marrow becomes destroyed or
fibrosed
In children, blood cells are actively produced in the marrow
cavities of all the bones.
By age 20, the marrow in the cavities of the long
bones, except for the upper humerus and femur, has become
inactive .
Active cellular marrow is called red marrow; inactive marrow
that is infiltrated with fat is called yellow marrow.

The bone marrow is actually one of the largest organs in the
body, approaching the size and weight of the liver. It is also
one of the most active.
Normally, 75% of the cells in the marrow belong to the white
blood cell-producing myeloid series and only 25% are
maturing red cells, even though there are over 500 times as
many red cells in the circulation as there are white cells.
This difference in the marrow reflects the fact that the
average life span of white cells is short, whereas that of red
cells is long.

STEM CELLS
• These cells have extensive proliferative capacity and
also the:
– Ability to give rise to new stem cells (Self Renewal)
– Ability to differentiate into any blood cells lines
(Pluripotency)
• They grow and develop in the bone marrow.
• The bone marrow & spleen form a supporting system,
called the
• “hemopoietic microenvironment”

Stem cells
• Totipotential stem cells- convert into any tissue type
• Pluripotent stem cell- Pluripotent hematopoeitic
stem cell
• Committed stem cells- CFU E, CFU G, CFU M, etc

CLONAL HEMOPOIESIS
PLURIPOTENT STEM CELL
STEM CELL
MULTIPLICATION COMMITTMENT
COMMITTED
STEM CELL
COMMITTED
STEM CELL
MULTIPLICATION
PROGENITOR
CELL
CFU: COLONY
FORMING UNIT

Hematopoietic stem cells (HSCs) are bone
marrow cells that are capable of producing all
types of blood cells.
They differentiate into one or another type of
committed stem cells (progenitor cells). These in
turn form the various differentiated types of blood
cells.
There are separate pools of progenitor cells for
megakaryocytes, lymphocytes, erythrocytes, eosi
nophils, and basophils; neutrophils and
monocytes arise from a common precursor.

PROGENITOR CELLS
• Committed stem cells lose their capacity for self-
renewal.
• They become irreversibly committed.
• These cells are termed as “Progenitor cells”
• They are regulated by certain hormones or substances
so that they can:
– Proliferate
– Undergo Maturation.

ERYTHROPOIESIS
15-20µm- basophilic cytoplasm,
nucleus with nucleoli.
14-17µm-mitosis, basophilic
cytoplasm, nucleoli disappears.
10-15µm-’POLYCHROMASIA’
Hb appears, nucleus condenses.
7-10µm- PYKNOTIC Nucleus.
Extrusion, Hb is maximum.
7.3µm- Reticulum of basophilic
material in the cytoplasm.
7.2µm- Mature red cell with Hb.

Pronormoblast
•15-20 microns
•Mitosis present
•Nucleus with multiple
nucleoli
•Basophilic cytoplasm
with polyribosomes
•No hemoglobin

Basophilic erythroblast
• Large nucleus
• Basophilic
cytoplasm
• Active mitosis
• Slight reduction in
size

Polychromatophilic erythroblast
• Chromatin lumps
• Hb starts appearing
• Reduced mitoses

Orthochromatic erythroblast
• Small and pyknotic
nucleus
• Eosinophilic
cytoplasm
• Mitoses absent

Reticulocyte
• Reticular nuclear
fragments
• Nucleus extruded
• Slightly larger than
RBCs

Reticulocytes
Young erythrocytes
Contain a short network of clumped ribosomes and RER.
Enter the blood stream
Fully mature with in 2 days as their contents are
degraded by intracellular enzymes.
Count = 1-2% of red cells
Provide an index of rate of RBC formation

Proerythroblast
or
pronormoblast
Basophilic
erythroblast
or
Early
Normoblast
Polychromatophilic
(or intermediate)
Erythroblast or
Normoblast
Dividing
Polychromatophilic
Erythroblast or
Normoblast
Orthochromatic
(Acidophilic)
erythroblast
Or
Late
Erythroblast
Orthochromatic
erythroblast
Extruding
Nucleus
Reticulocyte
Reticulocyte
(brilliant cresyl
blue dye)

Duration
Differentiation phase- from pronormoblast to
reticulocyte phase- 5 days
Maturation phase: from reticulocyte to RBC- 2
days

Factor needed of Erythropoiesis
1. Erythropoietin ( Released in response to Hypoxia)
2. Vitamin B 6 (Pyridoxine)
3. Vitamin B 9 (Folic Acid)
4. Vitamin B 12 (Cobolamin)
Essential for DNA synthesis and RBC
maturation
5. Vitamin C  Helps in iron absorption (Fe+++ 
Fe++)
6. Proteins  Amino Acids for globin synthesis
7. Iron & copper  Heme synthesis
8. Intrinsic factor  Absorption of Vit B 12
9. Hormones

Hormonal factors:
Androgens: increase erythropoiesis by stimulating the production of
erythropoietin from kidney.
Thyroid hormones:
 Stimulate the metabolism of all body cells including the bone marrow cells,
thus, increasing erythropoiesis.
 Hypothyroidism is associated with anemia while hyperthyroidism is
associated with polycythaemia.
Glucocorticoids:
 Stimulate the general metabolism and also stimulate the bone
marrow to produce more RBCs.
 In Addison’s disease (hypofunction of adrenal cortex) anemia
present, while in Cushing’s disease (hyperfunction of adrenal cortex)
polycythaemia present.

Hormonal factors
Pituitary gland: Affects erythropoiesis both directly
and indirectly through the action of several
hormone.
Haematopoietic growth factors: Are secreted by
lymphocytes, monocytes & macrophages to
regulate the proliferation and differentiation of
proginator stem cells to produce blood cells.

State of liver & bone marrow
Liver - Healthy liver is essential for normal
erythropoiesis because the liver is the
main site for storage of vitamin B12 , folic
acid, iron & copper. In chronic liver
disease anemia occurs.
Bone marrow - When bone marrow is
destroyed by ionizing irradiation or drugs,
aplastic anemia occurs.

Regulation of erythropoiesis
Tissue Oxygenation

Erythropoietin
• Glycoprotein with 165 amino acids, 4
oligosaccharide chains and molecular weight of 34,000
• Production- 85% by peritubular capillary bed interstitial
cells(Kidney) and 15% by perivenous hepatocytes( Liver)
• Also seen in brain, salivary glands, uterus, oviducts
• Site of Action: BONE Marrow

Factors increasing erythropoietin secretion:
(i) Hypoxia
(ii) Androgens
(iii) Growth Hormone
(iv) Catecholamines
(v) Prostaglandins
Factors inhibiting erythropoietin secretion:
(i) Estrogen
(ii) Theophylline

Action of Erythropoietin:
1. Formation of Pronormoblast from stem cell
2. Speeds up the differentiation through various
stages of erythropoiesis
Mechanism of Action:
• Formation of ALA synthetase
• Activation of Adenylyl Cyclase
• Synthesis of transferrin receptors

Maturation factors
Vitamin B12 and Folic acid:
– Essential for DNA synthesis (Thymidine triphosphate)
– Abnormal and diminished DNA
– Failure of division and maturation
– Macrocytic / Megaloblastic anemia
Other factors
– Cobalt
– Copper
– Vitamin C

HEMOLYTIC ANEMIA
Hemolytic anemia = reduced red-cell life
span

HAEMOLYTIC ANAEMIAS
•The normal red cell life is 110-120 days after
which the senile cells are removed by bone
marrow and splenic macrophages.
•Reduced red cell survival leads to increased red
cell production due to erythropoietin drive that can
compensate for the reduced red cell life and
maintain a normal Hb level.
•The mean red cell life is affected by molecular
changes in either the red cell membrane or
haemoglobin.

•A haemolytic state exists when the in vivo survival
of the RBC is shortened.
•Anaemia occurs if the onset of haemolysis is
sudden with no time for marrow compensation or in
severe chronic haemolysis when the mean red cell
life is very short.
•The usual marrow response in acute hemolytic
anemia is reflected by a reticulocyte index of 2–3,
whereas in long-standing chronic hemolysis, the
increase in erythropoiesis is approximately 6-fold.

Correcting Retic Count
Retic Index = Retic % x Patient Hct
Normal Hct
Absolute Retic = Retic % x RBC/mm3
Retic Production Index = Retic Index
Days in circulation

CLINICAL FEATURES
Jaundice: generally mild and often not noticed by the patient.
Anaemia: recent onset = acquired
long-standing = possibly congenital.
Haemoglobinuria: intravascular haemolysis.
Urobilinogenuria: increased Hb catabolism.
Splenic pain: spenomegaly or splenic infarction.
Leg ulcers: intrinsic red cell disorders, e.g. sickle cell disease.
Skeletal hypertrophy: severe congenital haemolytic anaemias
and thalassaemias.

CLASSIFICATION OF HEMOLYTIC
ANEMIAS
The course of the
disease
acute chronic
The place of RBC
distraction
intravascular extravascular
The whence acquired inherited

Haemolytic anaemia
Intravascular vs. Extravascular
Intravascular
• red cells lyse in the
circulation and release
their products into the
plasma fraction.
• Anemia
• Decreased Haptoglobin
• Hemoglobinemia
• Hemoglobinuria
• Urine hemosiderin
• Increased LDH
Extravascular
• ingestion of red cells by
macrophages in the
liver, spleen and bone
marrow
• Little or no hemoglobin
escapes into the
circulation
• Anemia
• Decreased Haptoglobin
• Normal plasma
hemoglobin
• Increased LDHdrpankajyadav05@gmail.com

Evidence of Hemolysis
• Low RBC survival with chromium tagging
study
• Unconjugated bilirubin
• Plasma Hb
• Decreased serum haptoglobin

Evidence of Erythropoiesis
• Polychromasia
• Increased reticulocyte
• “Shift” macrocytosis
• Hypercelluar BM

HEMOLYTIC ANEMIA
• INTRACORPUSCULAR HEMOLYSIS
– Membrane Abnormalities
– Metabolic Abnormalities
– Hemoglobinopathies
• EXTRACORPUSCULAR HEMOLYSIS
– Nonimmune
– Immune

Membrane Defect
• Hereditary spherocytosis
• Hereditary elliptocytosis
• Hereditary pyropoikilocytosis
• PNH (sensitivity to complement lysis --
sugar water test, Ham’s test)
• Hereditary stomatocytosis (possibly Rh
null)

Metabolic Defect
(enzyme deficiency)
• G6PD deficiency
– Hexose
monophosphate shunt
– Most common RBC
enzyme defect, >50
variants
– X-linked
– Low glutathione due to
low NADPH
– Oxidative lysis, Heinz
bodies, spherocytic
– Primaquine, fava
beans
• Pyruvate kinase
deficiency
– Glycolysis
– Low RBC ATP level
– Non-spherocytic
• B12 and folate deficiency
– Macrocytic
– HJ bodies
• Hemoglobinopathies
– Poikilocytosis
– Abnormal Hb

Hemoglobin Abnormalities
• Unstable hemoglobin disease
• Sickle cell anemia
• Other homozygous hemoglobinopathies
(CC, DD, EE; Chapter 52)
• Thalassemia major
• Hemoglobin H disease
• Doubly heterozygous disorders (such as
hemoglobin SC disease and sickle
thalassemia)

HEMOLYTIC ANEMIA - IMMUNE
• Drug-Related Hemolysis
PENICILLIN,CEFTRIAXONE,CEFOTETAN,QUINIDINE,ALPHA-
METHYLDOPA,LEVODOPA,PROCAINAMIDE,SULFA DRUGS
• Alloimmune Hemolysis
– Hemolytic Transfusion Reaction
– Hemolytic Disease of the Newborn
• Autoimmune Hemolysis
– Warm Autoimmune (WAIHA)70-80%
– Cold Autoimmune (CAIHA) 20-30%
– Mixed 7-8%
– Paroxysmal Cold Hemoglobinuria - raredrpankajyadav05@gmail.com

Warm vs. Cold Auto
WARM
• Reacts at 37 degC
• Insidious to acute
• Anemia severe
• Fever, jaundice frequent
• Intravascular not common
• Splenomegaly
• Hematomegaly
• Adenopathy
• None of these
COLD
• Reacts at room
temperature
• Often chronic anemia
• 9-12 g/dL (less severe)
• Autoagglutination
• Hemoglobinuria,
acrocyanosis and
raynaud’s with cold
exposure
• No organomegaly

EXTRACELLULAR DEFECTS
• Fragmentation Hemolysis
– DIC, TTP, HUS
– Extracorporeal membrane oxygenation
– Prosthetic heart valve
– Burns—thermal injury
– Hypersplenism
– Venom - Snake, Spider, Bee

Plasma Factors
• Liver disease (Spur-cell )
• Hypophosphatemia
• Vitamin E deficiency in newborns
• Abetalipoproteinemia
• Infections
– Malaria
– Babesia
– Clostridium
– Gram negative endotoxin
• Wilson Diseasedrpankajyadav05@gmail.com

Etiologic and Pathogenetic
Classification of the Hemolytic
Disorders
I. Inherited Hemolytic Disorders
A. Defects in the erythrocyte membrane
1. Hereditary spherocytosis
D. Deficiencies of enzymes involved in the pentose phosphate
pathway and in glutathione metabolism
1. Glucose-6-phosphate dehydrogenase (G6PD)
E. Defects in globin structure and synthesis
1. Unstable hemoglobin disease
2. Sickle cell anemia
3. Other homozygous hemoglobinopathies (CC, DD, EE; Chapter
52)
4. Thalassemia major
5. Hemoglobin H disease
6. Doubly heterozygous disorders (such as hemoglobin SC disease
and sickle thalassemia)

Disorders
II. Acquired Hemoltyic Anemias
A. Nonimmune: due to
1. Traumatic and microangiographic hemolytic anemias
2. Infectious agents
3.Chemicals, drugs, and venoms
4. Physical agents
5. Hypophosphatemia
6. Spur-cell anemia in liver disease
7. Vitamin E deficiency in newborns

Disorders
B. Immunohemolytic anemias
1. Iso (allo) immune:
transfusion of incompatible blood
Hemolytic disease of the newborn
2. Heteroimmune:
Virus, bacterial infections, chemical, Drug-induced
3. Autoimmune hemolytic anemia
Idiopathic (the essential cause is unknown)
Secondary or symptomatic (in case of lymphoma, chronic lymphocytic leukemia,
Other malignant disease, Immune-deficiency states, Systemic lupus
erythematosus and other autoimmune disorders, Virus and
mycoplasma infections)
Autoimmune hemolytic anemia caused by warm-reactive antibodies (Coomb’s
positive).
Autoimmune hemolytic anemia caused by cold-reactive antibodies
Cold hemagglutinin disease
Paroxysmal cold hemoglobinuria

Disorders
C. Paroxysmal nocturnal hemoglobinuria

The Three Primary Measures
Measurement Normal
Range
A. RBC count (RCC) 4- 5.7 million
B. Hemoglobin 12 to 17
Hematocrit (PCV) 38 to 50
A x 3 = B x 3 = C - This is the rule of thumb
Check whether this holds good in a given result
If not -indicates micro or macrocytosis or
hypochro.

The Three Derived Indicies
Measurement Normal
Range
A. RCC 4 to 5.7
B. Hemoglobin 12 to 17
C. Hematocrit 38 to 50
MCV C ÷ A x 10
MCH B ÷ A x 10
MCHC (%) B ÷ C x 100drpankajyadav05@gmail.com

Hemolytic Anemia
Anemia of increased RBC destruction
– Normochromic, normocytic anemia
– Shortened RBC survival
– Reticulocytosis – due to ↑ RBC destruction
Will not be symptomatic until the RBC life
span is
reduced to 20 days – BM compensates 6
times

Findings in Hemolytic Anemia
Reticulocyte count and RPI Increased
Serum Unconjugated Bilirubin Increased
Serum LDH 1: LDH 2 Increased
Serum Haptoglobin Decreased
Urine Hemoglobin Present
Urine Hemosiderin Present
Urine Urobilinogen Increased
Cr 51 labeled RBC life span Decreaseddrpankajyadav05@gmail.com

drpankajyadav05@gmai
l.com
Tests to define
the cause of hemolysis
1. Hemoglobin electrophoresis
2. Hemoglobin A2 (βeta-Thalassemia trait)
3. RBC enzymes (G6PD, PK, etc)
4. Direct & indirect antiglobulin tests
(immune)
5. Cold agglutinins
6. Osmotic fragility (spherocytosis)
7. Acid hemolysis test (PNH)
8. Clotting profile (DIC)

Peripheral smear

spherocytes
- hereditary spherocytosis
- acquired hemolytic
anemia (e.g. AIHA)
- physical or chemical
injury
- heat

elliptocytes
- heredirary elliptocytosis
- iron def. anemia
- myelofibrosis with
myeloid metaplasia
- megaloblastic anemia
- sickle cell anemia
- normal (<10% of cells)

Elliptocytosis

Stomatocytes
Slit like central pallor in RBC
1. Liver Disease
2. Acute
Alcoholism
3. H Stomatocyosis
4. Malignancies

Stomatocytes

acanthocytes
(irregular surface spicules)
irregularly spiculated
cells
with bulbous/rounded
ends of spicules
- abetalipoproteinemia
- liver disease

echinocytes
(crenated cells, burr cells)
regularly contracted cells
with smooth surface
undulation
- uremia
- artifact
- hyperosmolarity
- discocyte-echinocyte
transformation
(may be associated with reduced
ATP of RBCs)drpankajyadav05@gmail.com

Echinocytes
Evenly distributed spicules > 10
1. Uremia
2. Peptic ulcer
3. Gastric Ca
4. PK-D
Called Burr Cells

bite cells
Removal (“bites”) of
membrane by splenic
macrophages
- G6PD deficiency

Heinz bodies
denatured hemoglobin
- G6PD deficiency

Heinz body preparation with Crystal violet
Unstable hemoglobin

drepanocytes
(sickle cells)
- sickle cell anemia

basophilic stippling
irregular basophilic granules
(remnants of RNA)
fine stippling:
•increased production of RBCs
(reticulocytosis)
coarse stippling:
•lead poisoning
•impaired Hgb syntheisis
•megaloblastic anemia
•other sever anemias

dacrocytes
(teardrop cells)
- thalassemia
- myelofibrosis

leptocytes
(target cells)
- liver disease
(obstructive jaundice)
- post splenectomy
- hemoglobinopathies
(hypochromic anemias)
thalassemia
Hgb C disease
Hgb H diseasebeta thalassemia
relative increase of cell membrane --> ―target‖
formation drpankajyadav05@gmail.com

rouleaux
lined up RBCs in a row
- multiple myeloma

sideroblast/siderocyte
inorganic iron-containing
granules (Pappenheimer
bodies)
- sideroblastic anemia
abnormally trapped iron in
mitochondria forming a ring around
nucleus
- post splenectomy
ring
sideroblasts
intermediate
sideroblast
siderocyte

Howell-Jolly body
remnant of nuclear chromatin
single:
•hemolytic anemia
•post splenectomy
multiple:
•other abnormal erythropoiesis

Acanthocytes
5-8 spikes of varying length, irregular
intervals
Called Spur Cells, Occur in A H

schistocytes
(cell fragments)
indication of hemolysis
- megaloblastic anemia
- severe burns
- traumatic hemolysis
- microangiopathic
hemolytic anemia
(helmet cells, triangular cells)“helmet cell”

Shistocytes
1. MAHA
2. Prosthetic
valves
3. Uremia
4. Malignant HT
Fragmented, Helmet or triangle shaped
RBC

MAHA
Micro Angiopathic Hemolytic
Anemia

TRAUMATIC HEMOLYSIS

Malaria
Schistocytes

Normal BM High Power
E : G = 1 : 3

Bone marrow

Hyperactive BM – Skull
Hemolytic Anemia

Hemolytic anemia

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