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

 Definition of Terms:

• Microcytosis- refers to red cells that are small

• Macrocytosis- refers to red cells that are large

• Mean Corpuscular Volume (MCV)-

• Mean Corpuscular Hemoglobin Concentration

(MCHC)
 Definition of Terms:

• Mean Cellular Hemoglobin (MCH)

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

Orthochromatophilic
Reticulocyte Mature RBC
erythroblast
Life Span of RBC

• 90- 120 days

 Immediate Precursor of Red Blood Cell

• Reticulocyte

– Normal value: 0.5 to 1.5%

 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

• Hypochromatic – pale RBCs

 Ways to diagnose iron deficiency anemia?

2. Serum ferritin
– will be decreased. Careful, it’ll also be low in
diseased/ill patients (an acute phase reactant)

4. Bone Marrow
– staining

6. Treatment
– iron
 Causes of Macrocytic Anemia

• B12 deficiency

• Folate deficiency
Uses of B12 and Folate

• DNA synthesis

– B12 = co-factor

– Folate = transfer single carbon groups

 How do we get 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)
What do you see in the RBCs below?
How would we quantitate this?

• Anisocytosis refers to
red cells which vary
widely in size.
• The RDW
mathematically
measures the range
of red cell sizes.
What do you see in the RBCs below?
What diseases might they be associated with?

• Microcytosis refers to
red cells that are small.
• You can use the
lymphocyte nucleus as a
visual reference, or you
can use the MCV
• Associated with
– Iron deficiency
– Thalassemias
– Sideroblastic anemia
What do you see in the top
slide?
Characterizes what
diseases?

• Macrocytosis refers to large

red cells.
• Associated with
– Elevated reticulocyte count
– B12/folate deficiency
– Liver disease
– Thyroid disease
– Chemotherapy
– Anti-retrovirals (AZT)
What’s wrong with these RBCs?
Measured how? A likely cause?
• 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
What do you see on this slide?

• Poikilocytosis refers to
red cells that vary
widely in shape.

• Remember that
anisocytosis refers to
red cells that vary
widely in size.
What do you see here? Diseases?

• Target cells look like

bulls-eyes.
• Associated with
– Liver disease
– Thalassemias
– Hemoglobin C
– After splenectomy
What do you see here? Diseases?

• 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)
What do you see here? Diseases?

• Schistocytes are red

cell fragments with
sharp edges.
• They are a hallmark
of Microangiopathic
Hemolytic Anemia
(MAHA)
What do you see here?

• Sickle Cells are seen

in sickle cell anemia.
• Notice that this slide
has target cells as
well as a sickled cell.
What RBCs are here?
How do you distinguish the
two? Associated disease?

• 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.
What do you see here?
What causes it?

• Teardrop cells
• Seen in myelophthisic
processes, or
diseases of marrow
infiltration.
• Deformed as it tries to
squeeze out of the
bone marrow
• Howell-Jolly bodies
– are peripheral, small, round, purple inclusions
within red cells that represent nuclear remnants.

• They are seen after splenectomy, or in

cases of splenic hypofunction.
• Rouleaux are linear arrangements of red cells typically
described as “piles of coins on a plate”

• They are typically seen in disorders with increased

levels of immunoglobulin, such as Multiple Myeloma or
Waldenstrom’s macroglobulinemia.

• Severe hypo-albuminemia can also lead to reouleux

formation
• Red cell agglutination occurs when the red
cells are coated with IgM. IgM is large
enough to bridge two red cells and cause
agglutination.

• Unlike rouleaux, the red cell clumps are not

orderly and linear.
 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
Post-splenectomy blood findings
• Howell-Jolly bodies
– small round blue DNA remnants in periphery of RBCs

• Red cell abnormalities

– target cells, acanthocytes, schistocytes, NRBCs
Hemolytic Anemia
 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
(and the circulation does NOT like this!)
 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

• Acquired
– Immune-mediated
– Non-immune-mediated
• Most common defect leading to anemia?
– Hereditary spherocytosis

• Frequency?
– Affects 1/5000 Europeans

• Transmission?
– Autosomal dominant
• 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.

• Diagnosing?
– Increased osmotic fragility

• Treatment?
– Supplemental folate
– Splenectomy (but carefully consider timing in children)
• Functions of GP6D?
• Detoxification of metabolites of oxidative stress
• Elimination of methemoglobin

• Important Products of GP6D?

• NADPH
• Reduced glutathione
• Diagnostic methemoglobin precipitate?
• Heinz bodies
• Causes the formation of bite/blister cells

• Epidemiology of GP6D Deficiency?

• Type B is more prevalent
• Type A is in 20% of healthy Africans
• In 10-14% of African American men
• Also prevalent in the Mediterranean
• X-linked
 G6PD Deficiency: Agents to avoid

For SKAND…

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

Blister cell
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. You’re adding
patient RBCs!

• 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. You’re adding
patient serum!
 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
– First line is corticosteroids (i.e. prednisone).
– If steroids fail to work, or if patient relapses after
steroid taper, splenectomy may be necessary.
– Immunosuppressives such as cyclophosphamide
(Cytoxan) or azathioprine (Immuran) may be required
as third-line therapy.
 Warm-Antibody Hemolytic Anemias
Treatment

• Folate repletion

• Transfusion – determining factors:

– Heart failure, shock?
– Inadequate reticulocyte count?
 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
– You don’t need the drug in the body any more to get the
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

• Direct Coomb’s test + for C3, but not IgM!

• 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 (big difference

from warm antibody-mediated hemolysis)
 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

“BAr CoIns”
– Severe Burns
– Arsenic
– Copper
– Insect and spider bites
 Infections Causing Hemolysis

• Malaroa

• 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
 Normal Human Hemoglobins

• Gower Hemoglobin (Embryonic)

α2ε2

• Fetal Hemoglobin (HbF)

α2γ2

• Major Adult Hemoglobin (HbA)

α2β2

• Minor Adult Hemoglobin (HbA2)

α2δ2
 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

• PBG 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
 Hemoglobin M Diseases

• 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
 The Sickle Cell Diseases:
Inheritance, Appearance of Symptoms,
Diagnosis
• 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
 The Sickle Cell Diseases:
Inheritance, Appearance of Symptoms,
Diagnosis
• Compound heterozygotes (e.g., SC or S-β
Thalassemia) generally express a significant sickle
cell disease

• We 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
 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
 Megaloblastic
Anemia
• Red cells are
macrocytic
• Hypersegmented
neutrophils can be
seen
• Vitamin B12 or folate
deficiency
 Sickle Cell Anemia

Target Cell

Sickled Cell
 Myeloproliferative Diseases

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

 Polycythemia vera

Natural History – 4 phases:

2. Latent phase - asymptomatic
3. 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 of Polycythemia Vera

• Those common to ALL erythrocytosis

– Headache
– Decreased mental acuity
– Weakness
• Pruritis after bathing
• Hypermetabolic sxs
• Erythromelalgia
• Thrombosis
• Hemorrhage
 Symptoms of Polycythemia Vera

• 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
 P vera - 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
 P vera - 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
 P vera - 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