Red Blood Cell Disorders

PETER S. AZNAR, MD, FPSP

 3 Formed Elements of Blood

• Red Blood Cells (RBC) Erythrocytes

• White Blood Cells (WBC) Leukocytes

• Platelets Thrombocytes
 Functions of the Three Formed Elements

• RBC- carries oxygen

• WBC- responsible for the immune system and

prevention of infection

• Platelets- coagulation
 Definition of Terms:

• Ferritin- storage iron

• Transferrin- transport iron

• Total Iron Binding Capacity (TIBC)- test which

measures the extent to which iron-binding sites
in the serum can be saturated

• Erythropoiesis- is the process by which red blood

cells (erythrocytes) are produced
 Definition of Terms:

• Rouleaux formation- artificial stacking of red blood

cells

• Reticulocytes- immediate precursor of a mature

red blood cell

• Anisocytosis- variation in RBC size

• Poikilocytosis- variation in RBC shape

Rouleaux formation
 Definition of Terms:
• Microcytosis- refers to red cells that are small

• Macrocytosis- refers to red cells that are large

• Mean Corpuscular Volume (MCV)- refers to the mean volume of

all the erythrocytes

MCV = Hct x 10/ RBC count

NV: 80- 100fL

• Mean Cellular Hemoglobin (MCH)- refers to the mean mass

of hemoglobin in the RBC.

MCH = Hgb x 10 / RBC count

NV: 27-31 pg
 Definition of Terms:

• Mean Corpuscular Hemoglobin Concentration (MCHC)-

refers to mean concentration of hemoglobin in the red
cell

MCHC = Hgb / Hct

NV: 32-36 g/dL

• Hypochromasia- refers to red cells that have too little

hemoglobin

• Howell-Jolly bodies- are peripheral, small, round, purple

inclusions within red cells that represent nuclear
remnants
Howell Jolly Bodies
Life Span of RBC

• 90- 120 days

Order of Red Blood Cell Maturation
 Erythropoiesis
Polychromatophilic
Proerythroblast Basophilic erythroblast erythroblast

Orthochromatophilic
Reticulocyte Mature RBC
erythroblast
 Immediate Precursor of Red Blood Cell

• Reticulocyte

– Normal value: 0.5 to 1.5%

 Classification of Anemias Based on MCV
Anemia
Males: Hb<13.5 Hct <41
Females: Hb <12 Hct<36

MCV

MCV<80 MCV 80-100 MCV>100

-Iron Deficiency Reticulocyte Count -Megaloblastic Anemia

- Thalassemia B12/ Folate Deficiency

-AOCD - Alcoholic Liver Disease
- Sideroblastic Anemia Low High

- Marrow Failure - Sickle Cell Anemia

- Aplastic Anemia - G6PD Deficiency
- Myelofibrosis - Hereditary Spherocytosis
- Leukemia/Metastasis - AIHA
- Renal Failure - PNH
- Anemia of Chronic Disease
 Causes of Iron Deficiency

1. Inadequate intake

3. Malabsorption

5. Diversion of iron during pregnancy

7. Blood loss
 RBC Characteristics in Iron Deficiency
Anemia?

• Microcytic – small RBCs

• Hypochromic – pale RBCs

 Ways to diagnose iron deficiency anemia

2. Serum ferritin
– will be decreased

3. Bone Marrow
– staining

5. Treatment
– iron
 Iron Panel for Microcytic Anemias

Iron Deficiency AOCD Thalassemia Sideroblastic

Minor Anemia
Serum iron Decreased Decreased Normal Increased

TIBC Increased Decreased Normal Decreased

% Saturation Decreased Decreased Normal Increased

Serum Ferritin Decreased Increased Normal Increased

 Causes of Macrocytic Anemia

• B12 deficiency

• Folate deficiency
Uses of B12 and Folate

• DNA synthesis

– B12 = co-factor

– Folate = transfer single carbon groups

 Sources of Folate and B12

• Folate
– In leafy green veggies, liver, yeast
– Destroyed by cooking
– Need 100-200 micrograms daily

• Vitamin B12
– In animal products
– Unaffected by cooking
– Need 1-2 micrograms daily
 Folate Deficiency – 3 major causes

• Dietary

• Malabsorption

• Increased usage
 3 Ways to Diagnose Folate Deficiency

2. Morphology
– macrocytic RBCs and hypersegmented
neutrophils

3. Serum folate

5. Red cell folate

Megaloblastic Anemia
Possible Causes?
B12 or folate deficiency
 B12 Deficiency – 3 major causes

2. Pernicious Anemia

4. Pancreatic Insufficiency

6. Malabsorption
 3 Ways to Diagnose- B12 Deficiency

• Morphology

• Serum B12

• Neurologic findings
– Demyelination of spinal cord, cerebral cortex
 Treating B12 & Folate Deficiencies

• B12
– IM B12 supplementation for life

• Folate
– Daily folate supplement (1mg/day)
 Megaloblastic
Anemia
• Red cells are
macrocytic
• Hypersegmented
neutrophils can be
seen
• Vitamin B12 or folate
deficiency
What do you see in the RBCs below?
How would we quantitate this?

• Anisocytosis refers to
red cells which vary
widely in size.
• Microcytosis refers
to red cells that are
small.
• Associated with
– Iron deficiency
– Thalassemias
– Sideroblastic anemia
• Macrocytosis refers to large
red cells.
• Associated with
– B12/folate deficiency
– Liver disease
– Thyroid disease
– Chemotherapy
– Anti-retrovirals (AZT)
• Hypochromasia refers
to red cells that have
too little hemoglobin.
• The area of central
pallor is more than 1/3
the total red cell
diameter.
• This is measured by the
MCH (mean cellular
hemoglobin)
• Iron deficiency
• Poikilocytosis refers to
red cells that vary
widely in shape.
• Target cells look like
bulls-eyes.
• Associated with
– Liver disease
– Thalassemias
– Hemoglobin C
– After splenectomy
• Spherocytes have a loss
of central pallor.
• Can be seen in
– Hereditary spherocytosis
– Autoimmune hemolysis
• If due to autoimmune
hemolysis, the cells are
smaller (i.e.
microspherocytes)
• Schistocytes are red
cell fragments with
sharp edges.
• They are a hallmark
of Microangiopathic
Hemolytic Anemia
(MAHA)
• Sickle Cells are seen
in sickle cell anemia.
• Notice that this slide
has target cells as
well as a sickled cell.
• Echinocytes, or burr cells,
have small, regular
projections. Seen in renal
disease

• Acanthocytes, or spur
cells, have larger, irregular
projections, and are seen
in liver disease.
• Teardrop cells
• Seen in myelophthisic
processes, or
diseases of marrow
infiltration.
• Deformed as it tries to
squeeze out of the
bone marrow
Hemolytic Anemia
 General Clinical Features of Hemolytic
Anemias
• Splenomegaly is generally present
• Patients have an increased incidence of pigmented
gallstones.
• Dark urine (tea-colored or red), jaundice, scleral
icterus
• Patients may have chronic ankle ulcers.
• Aplastic crises associated with Parvovirus B19, may
occur
• Increased requirement for folate
 Sites of Red Cell Destruction

• Extravascular Hemolysis
– Macrophages in spleen, liver, and marrow
remove damaged or antibody-coated red cells

• Intravascular Hemolysis
– Red cells rupture within the vasculature,
releasing free hemoglobin into the circulation
 Evidence for Increased Red Cell Production

• In the blood:
– Elevated reticulocyte count
– May be associated with high MCV
– Circulating NRBCs may be present
• In the bone marrow:
– erythroid hyperplasia
– reduced M/E (myeloid/erythroid) ratio
• In the bone:
– Deforming changes in the skull and long bones
(“frontal bossing”)
 Evidence for Increased Red Cell Destruction
• Biochemical consequences of hemolysis in general
• Elevated LDH
• Elevated unconjugated bilirubin  jaundice, scleral icterus
• Lower serum haptoglobin
• Hemoglobinemia
• Hemoglobinuria
• Hemosiderinuria
• Morphologic evidence of red cell damage
• Schistocytes
• Spherocytes
• Bite/blister cells
• Reduced red cell life-span
 Classification by Etiology

• Congenital
– Defects in membrane skeleton proteins
– Defects in enzymes involved in energy
production
– Hemoglobin defects (hemoglobinopathies)

• Acquired
– Immune-mediated
– Non-immune-mediated
 Hereditary Spherocytosis

• Frequency
– Affects 1/5000 Europeans

• Transmission
– Autosomal dominant
 Hereditary Spherocytosis
• Pathophysiology
– Defect is in proteins of the membrane skeleton, usually
spectrin or ankyrin
– Lipid microvesicles are pinched off in the spleen and other
RE organs, causing decreased MCV and spherocytic
change.

• Diagnostic Test
– Increased osmotic fragility
– Normal MCH with increased MCHC

• Treatment
– Supplemental folate
– Splenectomy
 Glucose-6-Phosphate Dehydrogenase
deficiency (G6PD deficiency)

• Functions
• Detoxification of metabolites of oxidative stress
• Elimination of methemoglobin

• Important Products
• NADPH
• Reduced antioxidant glutathione
 Glucose-6-Phosphate Dehydrogenase
deficiency (G6PD deficiency)

• Diagnosis
• methemoglobin precipitate
• Heinz bodies
• Causes the formation of bite/blister cells

• Epidemiology
• Type A- is in 20% of healthy Africans
• Also prevalent in the Mediterranean
• X-linked
 G6PD Deficiency: Agents to avoid

– Fava beans (specially in Mediterranean type)

– Sulfa drugs
– Vitamin K
– Anti-malarials
– Naphtha compounds (mothballs)
– Dapsone
G6PD Deficiency

Blister cell
 Paroxysmal Nocturnal Hemoglobinuria (PNH)

a. Abnormality
• Decreased glycosyl phosphatidyl inositol (GPI)-
linked proteins, especially decay accelerating factor
(DAF):

• The function of DAF is to inhibit the activation of the

complement cascade by breaking down C3 convertase

• Deficiency of DAF results in increased complement activity

• All cells in blood have increased sensitivity to the lytic

actions of complements
a) Symptoms are episodes (paroxysms) of hemolysis
at night.

c) Acidosis in vivo, which occurs during sleep

(breathing slowly retains CO2) and exercise (lactic
acidosis), causes activation of complement.

e) PNH is a clonal stem cell disorder that therefore

affects all cell lines.

g) Pancytopenia in peripheral blood: anemia,

leukopenia, thrombocytopenia

i) Complications: increased risk for aplastic anemia,

leukemia, and venous thrombosis
a) Lab tests for PNH
i. Sucrose in vitro (sucrose lysis test)
ii. Acidosis in vitro (Ham’s test)
How will you diagnose an autoimmune
cause of hemolytic anemia?
 Coomb’s Test

• The Direct Coomb’s

– DAT (Direct Antiglobulin Test) - tests for IgG or C3
DIRECTLY ON THE RED CELLS.

• The Indirect Coomb’s

– tests for IgG or C3 in the serum which react with
generic normal red cells. This is also known as the
antibody screen in blood-banking.
 Warm-Antibody Hemolytic Anemias
Clinical Features

• Splenomegaly, jaundice usually present.

• Depending on degree of anemia and rate of fall in
hemoglobin, patients can have VERY
symptomatic anemia
• Lab Dx -
↑reticulocytes, ↑ bili, ↑ LDH,
– positive Coomb’s test - both direct and indirect.
– SPHEROCYTES are seen on the peripheral
smear.
 Warm-Antibody Hemolytic Anemias
Treatment

• Immunosuppressive Treatment
– Corticosteroids (i.e. prednisone).
– Splenectomy may be necessary.
– Immunosuppressives such as
cyclophosphamide (Cytoxan) or azathioprine
(Immuran) may be required as third-line
therapy.
 Drug-Induced Immune Hemolysis
Three general mechanisms

• Innocent bystander
– the Ab was directed at the drug, but it cross
reacted w/ RBCs
– Drug must be present for hemolysis to occur
– Quinine, Quinidine, Isoniazide

• Hapten
– Drug binding to RBC  Abs that react to this complex
– Penicillins, Cephalosporins
 Drug-Induced Immune Hemolysis
Three general mechanisms

• True autoimmune
– no drug needed in the body any more to produce
hemolysis
– Alpha-methyldopa, L-DOPA, Procainamide
 Cold Agglutinin Disease

• IgM antibodies bind to I antigens of RBCs when cold (falls

off when warm)

• Causes agglutination  cyanosis & ischemia of

extremities

• Has both intravascular and extravascular hemolytic

components
 Cold Agglutinin Disease

• Primary, or associated w/ Mycoplasma,

Mononucleosis, or lymphoproliferative disease

• Treat by avoiding cold & folate repletion

• Corticosteroid and splenectomies uneffective

 Non-Immune Hemolytic Anemia
Classification

• Mechanical trauma to red cells

– Microangiopathic Hemolytic Anemia
– Abnormalities in heart and large vessels
– March Hemoglobinuria

• Infections

• Drugs, Chemicals, and Venoms

 Chemical & Physical Agents Causing
Hemolysis
– Severe Burns
– Arsenic
– Copper
– Insect and spider bites
 Infections Causing Hemolysis

• Malaria

• Babesia microti

• Clostridium welchii

• Bartonella bacilliformis
 Basic Structure of All Human Hemoglobin

• Each hemoglobin molecule is composed of:

– 4 iron-containing, tetrapyrrole heme rings
– 4 polypeptide globin chains
• 2 alpha chains
• 2 non-alpha chains

• Each α globin chain has 141 amino acids

• All non-α chains have 146 amino acids
• There is considerable structural homology among the
non-alpha chains
Hemoglobinopathies
 Normal Human Hemoglobins

• Gower Hemoglobin (Embryonic)

α2ε2

• Fetal Hemoglobin (HbF)*

α2γ2

• Major Adult Hemoglobin (HbA)

α2β2

• Minor Adult Hemoglobin (HbA2)

α2by
* Detected δ2 Kleihauer-Betke and Apt test
 Heme Synthesis

• Begins with condensation of glycine & succinyl

Co-A  δ-amino levulinic acid (δ-ALA).
– The rate-limiting step in heme synthesis
– Requires intra-mitochondrial enzyme ALA-
synthase

∀ δ-ALA travels to cytoplasm; converted to

porphobilinogen (PBG), a monopyrrole.
 Heme Synthesis

• Porphobilinogen is converted from monopyrrole to

biologically active form protoporphyrin IX, a tetrapyrrole.

• Iron inserted into tetrapyrrole ring n the mitochondria

• Heme synthesis stimulated by iron & repressed when iron

is inadequate (e.g., iron deficiency)
 Location of the Globin Genes

• Genes for the non-α chains are located on

Chromosome 11. This is referred to as the β-
globin gene cluster
∀ α Chain genes are located on Chromosome 16
• There is duplication of the genes for:
∀ α Globin
∀ γ Globin (Gγ and Aγ) *
• G
γ and Aγ differ from one another only at position 136 where
they have glycine & alanine respectively
 Location of the Globin Genes

• Synthesis of the non-α chains involves a

coordinated switching that proceeds from
embryonic (ε) to fetal (γ) to adult (β) globin
chains
• Yolk sac (ε)  liver/spleen (γ)  marrow (β)
 Structure of the Hemoglobin Molecule

• Each Hb is comprised of 4 subunits: 2 identical α

chains & 2 identical non-α chains

• Each chain is arranged in the form of an α-helix with

8 individual helical segments (labeled A - H)

• Each globin molecule has both hydrophobic &

hydrophilic areas
 Structure of the Hemoglobin Molecule

• The iron-containing heme ring is buried within a

very hydrophobic region of the globin that is
called the “Heme Pocket”

• The hydrophobic nature of this region protects the

iron residue from oxidation, thereby maintaining
it in the active, reduced form

• Each iron atom in the center of the heme residue is

held in place and kept in the active, reduced Fe++
state by two histidine residues
 Possible Consequences of a
Hemoglobinopathy

• No detectable effect
• Instability of the hemoglobin molecule
• An increase or a decrease in oxygen affinity
• Inability to maintain the heme iron in its active,
reduced state (methemoglobinemia)
• Decreased solubility of the hemoglobin molecule
 Unstable Hemoglobinopathies

• Most of the unstable hemoglobinopathies involve a

mutation in the region of the heme pocket

• These mutations enable water to gain access to this

very hydrophobic region of the molecule

• The end result is heme instability, denaturation, and

release of heme from its binding site

• The demonstration of Heinz Bodies in these red cells

is evidence of the presence of an unstable
hemoglobin mutant
 Hemoglobinopathy Altering Oxygen
Affinity

• Increased Oxygen Affinity

– Stabilization of the Oxy conformation increases the

oxygen affinity of the hemoglobin molecule

– The presence of such an effect can be confirmed by

demonstrating a left shift in the Oxygen
Saturation Curve

– Individuals with an increase in oxygen affinity

typically exhibit erythrocytosis
 Hemoglobinopathy Altering Oxygen
Affinity

• Decreased Oxygen Affinity

– Stabilization of the Deoxy conformation produces a
decrease in the the oxygen affinity of
the hemoglobin molecule
– The presence of such an effect can be confirmed by
demonstrating a right shift in the
Oxygen Saturation Curve
– Individuals with a decrease in oxygen affinity are
typically somewhat anemic
 Causes of Hemoglobinopathies:

I. Structural defect
- Alterations in the gene for one of the two
hemoglobin subunit chains are called mutations.
eg. sickle hemoglobin

II. Diminished production

- eg. thalassemia
 Causes of Hemoglobinopathies:

III. Abnormal associations of otherwise normal

subunits
- A single subunit of the alpha chain (from the
a-globin locus) and a single subunit from the
b-globin locus combine to produce a normal
hemoglobin dimer. With severe a-thalassemia,
the b-globin subunits begin to associate into
groups of four (tetramers) due to the paucity of
potential a-chain partners. These tetramers of
b-globin subunits are functionally inactive and
do not transport oxygen
 Clinically Significant Variant Hemoglobins
Hemoglobinopathy Alpha Beta Comments

Hemoglobin S α2βS2 Normal Defective

Hemoglobin C α2βC2 Normal Defective Hemolytic anemia, splenomegaly,

target cells are characteristic
Hemoglobin E α2βΕ2 Normal Defective Benign, common in SE Asia

Hemoglobin Constant HCSpr Defective Normal Long alpha chain

Spring
Hemoglobin H β4 Absent Normal Paucity of alpha chains
Screening test: Heat instability test
Hemoglobin Barts β4 Absent Normal Not compatible with life
Hemoglobin M Normal Normal A group of abnormal hemoglobins
in which a single amino acid
substitution favors the
formation of methemoglobin.
 Thalassemia

• The thalassemias are a group of disorders in which the

normal hemoglobin protein is produced in lower amounts
than usual. The genes are defective in the amount of
hemoglobin they produce, but that which they produce
(generally) is normal. The thalassemias are a complex
group of disorders because of the genetics of hemoglobin
production and the structure of the hemoglobin molecule.

• A quantitative deficiency in RNA resulting in decreasing

globin chain production
 Hemoglobin M Disease

• The Hemoglobin M disorders are seen when a

substitution has occurred at the locus of either the
proximal or distal histidine
• Typically, this involves a his tyr substitution which then
forms an iron-phenolate complex
• Hemoglobin with its iron in the oxidized Fe+++ state is
incapable of binding oxygen
• This form of hemoglobin (called Methemoglobin) has a
brownish appearance
• Patients with Hemoglobin M disease are typically
cyanotic
 Sickle Cell Disease:

• The most common sickle cell disease (SCD) is

called sickle cell anemia (HbSS)
• However, there are a number of other SCD
genotypes - compound heterozygous states
• The sickle mutation is inherited in an autosomal
co-dominant fashion
• Individuals with sickle cell trait (AS) have roughly
equal amounts of HbA & HbS and are
generally asymptomatic
 Sickle Cell Disease:

• Compound heterozygotes (e.g., SC or S- β

Thalassemia) generally express a significant sickle
cell disease

• Dx with electrophoresis:
- Hb C has a positive; HbS is neutral, HB A is
negative.
- Movement: HbA > HbS > HbC
 Sickle Cell Anemia Pathophysiology
●
The presence of the abnormal (or sickle)
hemoglobin (HbS) within the cells of the
affected individuals
●
The decreased solubility & the tendency of
this abnormal hemoglobin to polymerize
when it assumes the deoxy conformation

● In HbS, the negatively charged glutamic acid

at β6 position is replaced by an uncharged
valine residue
 Sickle Cell Anemia Pathophysiology

● In deoxy conformation, the valine at β6 position

approaches the phenylalanine at β 85 position on
adjacent HbS molecule.

• Multiple critical contact points that enable the

hemoglobin molecules to attach to one another

• The polymer begins as a small nucleus of hemoglobin

molecules  aligned polymer with a total of 7 anti-
parallel pairs (or 14 individual hemoglobin chains)
 SICKLE CELL DISEASE
Clinical Features
– Painful vaso-occlusive crisis
– Strokes
– Retinopathy
– Acute chest syndrome
– Pulmonary hypertension
– Sickle cell nephropathy
– Biliary tract disease
– Leg ulcers
– Avascular necrosis of the large joints
– Aplastic crisis
 SICKLE CELL DISEASE
Therapeutic Approaches

• Reactivate Fetal Hemoglobin Production using

Hydroxyurea
• Chemical inhibition of Hb S polymerization
• Increase in intracellular hydration
• Altering RBC/Endothelial cell interactions
• Bone marrow transplantation
• Gene therapy
 Sickle Cell Anemia

• Screening test
– Dithionite solubility test
 Sickle Cell Anemia

Target Cell

Sickled Cell
 Myeloproliferative Diseases

• Includes:
• Polycythemia vera
• Essential Thrombocythemia
• Myelofibrosis
• Chronic Myelogenous Leukemia
Polycythemia Vera
 Polycythemia vera

• Most of cells in circulation are derived from a

single, neoplastic stem cell

• Does not need Epo to produce more cells

• Diagnosis based on low/absent levels of Epo

 4 Phases

1. Latent phase - asymptomatic

2. Proliferative phase -pts may have sxs of:
– Hypermetabolism
– Hyperviscosity
– Thrombosis
• Spent phase - ↓ red cell mass, anemia,
leukopenia, secondary myelofibrosis,
increasing HSM. 20% of pts
• Secondary AML
– 1-2% of pts treated with phlebotomy alone
 Symptoms

• Those common to all erythrocytosis

– Headache
– Decreased mental acuity
– Weakness
• Pruritis after bathing
• Hypermetabolic sxs
• Erythromelalgia
• Thrombosis
• Hemorrhage
 Signs

• PE findings
– Facial plethora
– Splenomegaly
– Hepatomegaly
– Retinal vein distension
• Lab findings
– Basophilia
– Low EPO levels
– Increased Hbg/HCT, WBCs, platelets, uric acid,
B12, leukocyte alkaline phosphatase score,
abnormal platelet function
 Treatment

• Phlebotomy
– Draw 500 cc blood 1-2x/wk to target Hct 45%;
maintain BP w/ saline
– Generally, the best initial treatment for P vera –
rapid onset
– Downsides:
• Increased risk of thrombosis
• No effect on progression to spent phase
• May be insufficient to control disease
 Treatment

• Myelosuppressive agents
– Hydroxyurea
• can be used in conjunction with phlebotomy
• May increase the risk of leukemic transformation from
1-2% to 4-5%
– 32P – kills some of the proliferating cells
• increase the risk of leukemic transformation from
1-2% to 11%
• Single injection may control hemoglobin and platelet
count for a year or more.
– Alkylating agents such as busulfan
 Treatment

• Interferon alpha
– Benefits
• No myelosuppression
• No increase in progression to AML
• No increase in thrombosis risk

– Drawbacks
• Must be given by injection up to daily
• Side effects may be intolerable in many pts: flu-like
symptoms, fatigue, fever, myalgias, malaise
THANK YOU!