Sickle Cell Anemia
(Hemoglobinopathies)
By: Jessica Hebert, Rob
McElroy, Beth McQuinn, Yuway
Tsing, Elias Kass, Bill Walter,
Amelia Kramer
What are hemolytic
anemias?
• Decreased life-span of
erythrocytes
• Removal of RBCs may
be:
– Intravascular (within the
blood)
– Extravascular (within the
spleen)
• Associated with:
– Increased erythropoiesis
– Increased hemoglobin
catabolism byproducts
Possibilities?
• Many types of hemolytic anemias:
– G6PD-Deficiency
– Hemoglobinopathies
• Sickle Cell Disease
• Hereditary Spherocytosis
• Thalassemias
– Immunohemolysis
– Others…
Background:
Prevalence in Populations
Population
Prevalence
African American
1/375 HbSS , 1/835 HbSC , 1/10 HbSA (trait)
American
> 50,000 (2 mil with sickle cell trait)
Mediterranean descent
significant
Caribbean descent
Not significant
East Indian Ancestry
Not significant
Arabian
Not significant
Latin American
1/1000-1400
Greeks
Not significant
Italians
Not significant
•http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Search&db=books&doptcmdl=GenBookHL&term=sickle+cell+prevalence+AND+
hstat%5Bbook%5D+AND+340265%5Buid%5D&rid=hstat6.section.17004
Clinical Presentation: Signs and Symptoms
General anemia
pallor, fatigue, SOB on exertion
Acute chest syndrome
cough, dyspnea, chest pain, fever
Infection
malaise, cough and chest pain, diarrhea and/or vomiting
Painful crises
persistent pain in skeleton, chest, and/or abdomen
Hand-foot syndrome
swollen and painful hands and feet (by 2 years of age: 50% of Jamaican
and 25% of American children with sickle cell anemia have experienced
at least one episode of dactylitis)
Stroke
(affects 10% of patients; 6-17% of children and young adults), sudden
neurologic deficits including motor, difficulty with language, writing, and/or
reading; seizures; sensory deficits; altered consciousness
Priapism
penile erection not related to arousal
Delayed growth and
puberty
Patient more slender or small in size.
Jaundice
Yellowing of skin and eyes
Other
Some people remain asymptomatic into late childhood and are only
incidentally diagnosed
http://www.ornl.gov/sci/techresources/Human_Genome/posters/chromosome/sca.shtml
Background
Clinical Presentation
• There are a range of
Treatments per extremes or
per symptoms
Sickle Cell Anemia
A hereditary
genetic disorder
that results in
the production
of abnormal
hemoglobins
In Sickle Cell, the Beta chain is affected
by a point mutation…
Source: Robbins
One small point mutation leads to a major problem
when HbS is deoxygenated…
Deoxyhemoglobin A (HbA)
Deoxyhemoglobin S (HbS)
http://www.ornl.gov/sci/techresources/Human_Genome/posters/chromosome/hbb.shtml
A prolonged environment of low oxygen leads to
aggregation and polymerization of hemoglobins into long
chains of rod-like fibers, causing the RBC to form the
shape of a crescent or sickle.
• Donut-shaped
• Soft and malleable
• Able to pass through
small spaces
• Lifespan ~120 days
• Sickle-shaped
• Hard and rod-like
• Gets stuck in narrow
spaces
• Lifespan ~20 days
http://www.humanillnesses.com/original/images/hdc_0001_0003_0_img0235.jpg
Sickling of red blood cells leads to:
1. Vaso-occlusive
complications
2. Severe anemia
3. Chronic
hyperbilirubinemia
Vaso-occlusive complications
Occur in areas with:
• Prolonged, low
oxygen tension
• Decreased pH
• Inflammation
• Low blood flow
Source: Robbins
Severe Anemia and Chronic
Hyperbilirubinemia
1. Young RBCs with HbS can
alternate between the
sickled and normal
formation.
2. However, sickling causes
oxidative damage to the
RBCs cell membrane (and
to the cells around it).
3. Eventually, RBCs become
irreversibly sickled.
Severe Anemia and Chronic
Hyperbilirubinemia
• Irreversibly sickled cells
end up in the spleen.
• Hemolysis occurs at
the splenic cords.
• Decreased RBCs and
increased bilirubin
• Infarction and fibrosis
causes
autosplenectomy.
Source: Robbins
G6PD-Deficiency Distribution
G6PD-Deficiency Distribution
• 12% of males of African descent (A type)
• 22% of Southeast Asian males (Mahedian
type)
• 50% of Kurdish males (Mediterranean type)
• Also, a significant prevalence in males of
Italian, Greek, Lebanese, Iraqi, and
Sephardic heritage (Mediterranean type)
G6PD-Deficiency
Presenting Symptoms
• Fatigue or muscle
weakness
• Jaundice or pallor
• Dyspnea
• Dark urine
• Pain (especially
abdominal)
• Splenomegaly
Glucose 6-Phosphate
G6PD-Deficiency – Pathology
• Genetic abnormalities (of which there are
several varieties) lead to deficient or
impaired enzyme function
• High levels of oxidants (from foods, drugs,
exercise, infections) may damage or
denature proteins within the erythrocyte
• RBCs may then be subject to intravascular
or extravascular hemolysis
G6PD Crisis Precipitation - Beans
• Broad beans (favism)
– Fava beans, bell beans, fever beans, hava
beans, pigeon beans, horse beans, tick
beans, silkworm beans, English dwarf beans
G6PD Crisis Precipitation Oxidizing Drugs
– Antimalarials
(primaquine, quinine,
quinidine)
– NSAIDs (aspirin,
ibuprofen)
– Antibiotics
(sulfonamides)
– Nitrofurantoin
– TCM herbs (rhizoma
coptidis, margarita,
calculus bovis)
Other oxidants
– Henna – primarily infants and pregnant
women
– Naphthlene (moth balls) – regular exposure
Early Lab Tests – CBC
•
•
•
•
•
•
•
RBC – Low
Hct – Low
Hb – Low
MCV – Normal to High
MCHC – Low to Normal
MCH – Low to Normal
So…initial CBC is only your starting point
Early Lab Tests – Urinalysis
• Hemoglobin
– Elevated post-crisis
• Bilirubin
– Elevated post-crisis
• Urobilinogen
– Elevated post-crisis
• May also see hematuria, proteinuria, LDH
• These account for dark color of urine following a
crisis, but are also non-specific for any given
type of hemolytic anemia
Early Lab Tests - Smear
• Often readily seen with initial CBC, or easily ordered
alongside
• If anemias are suspected, probably best to run a smear
in the early stages
• G6PD-Deficiency may show:
– Heinz bodies
• Denatured Hb bound to cell membrane
• May damage cell membrane, leading to intravascular hemolysis
– “Bite” cells
• Spleen removes portion of RBC that had Heinz body, preventing
intravascular hemolysis
– These aren’t typically present immediately following an acute
hemolytic crisis (problem cells have been destroyed)
Other Non-Specific Lab Tests
• Reticulocyte count
–
–
–
–
May be elevated following acute crisis (6-8 weeks)
May be normal in non-crisis situations
May be reduced in aplastic crisis (could go to zero)
Should be considered in conjunction with smear, other data
• Serum bilirubin
– Present in the unconjugated (prehepatic) form
– Elevated after an acute hemolytic crisis
• Serum haptoglobin
– Binds to Hb in the blood for removal by spleen (preventing Hb
from excretion in the urine, which could lead to renal failure)
– Decreased (depleted) after an acute hemolytic crisis
Screening for G6PD
• Cells from G6PD-deficient subjects can’t
convert the oxidized substrate to a
reduced state
• Different tests use different substrates
• Substrates can either be the enzyme’s
natural substrate (NADP) or substrates
from secondary reactions
Fluorescent Screening Test
• G6PD generates NADPH
• NADPH fluoresces under long-wave UV
light
• No G6PD? No fluorescence.
Fluorescent Screening Test
• Shortcomings:
– False Negative: young red blood cells have more
G6PD activity. During an acute hemolytic episode,
reticulocytosis results in a bigger population of young
red blood cells, so even in G6PD Deficiency, the
fluorescence may appear normal.
– False Positive: if the patient is anemic, very little
fluorescence may be seen because there are very
few red blood cells.
Methemoglobin Reduction Test
• Sodium nitrite converts Hb (hemoglobin) to
Hi (methemoglobin)
• Adding methylene blue should stimulate
the pentose phosphate pathway, reducing
methemoglobin
• In G6PD Deficiency, methemoglobin
persists
Methemoglobin Reduction Test
• Normal blood → clear red color
• Deficient blood → brown color
• Heterozygotes → intermediate color
Methemoglobin Reduction Test
- Takes longer than fluorescence
+ Extremely inexpensive
+ Requires only a water bath
• Test should be complemented by
cytochemical analysis
Cytochemical Analysis
• Only way to reliably detect deficiency in
heterozygous women
• More reliable than assay
• Good complement for other tests
Cytochemical Analysis
• Treat red cells to convert their oxyhemoglobin (HbO2) to
methemoglobin (Hi)
• In the presence of G6PD, Hi converts back to HbO2
• Add MTT, which will be reduced by HbO2 to an insoluble
form
• In G6PD Deficiency:
– Hi persists
– MTT is not reduced
– Ergo, cells are not stained
Cytochemical Analysis
Cytochemical Analysis
• Interpretation:
– Normal G6PD activity: majority stained
– Hemizygous: majority unstained
– Heterozygotes:
• Most show mosaicism (40-60% unstained)
• Some have only 2-3% unstained (fortunate
lyonization)
G6PD Assay
• Tracks activity of enzyme by following rate
of production of NADPH over time
• NADPH has peak of UV light absorption at
340nm
• Note potentially misleading results in
following table
G6PD Assay
Ma le
Fem a le
In he a lth
In in creas e d
de mo ly s is
u n relate d t o
G6P D
defi ci en c y
Dur in g
rec o ve ry f ro m
G6P D- re lat e d
an e m ia
Gd + (n orm al)
Gd ( e nti rely defici e nt )
Gd+/ Gd + ( norm al)
Gd -/ Gd( e nti rely defici e nt )
Gd+/ Gd ( so m e what
deficie nt)
7 -1 0
<2
2 -7
15
4
4 -9
6.5 ( a p pe ar s
norm al)
6 -1 0 ( a p pe a rs
norm al)
Values are examples only (Lewis 10e, p226)
G6PD Assay
• Interpretation:
– Red cells are likely to lyse from G6PD deficiency if
they have less than ~20% of normal enzyme activity
• Confounding factors:
– With reticulocytosis, the relatively large population of
reticulocytes yields a higher enzyme activity, covering
up deficiency
– Therefore, a low-normal enzyme activity assay during
reticulocytosis points towards G6PD deficiency
(without deficiency, values would be very high during
reticulocytosis)
– Re-evaluate after reticulocytosis slows
Choosing Appropriate Tests
• More than one test should be used
• Are they in the middle of a hemolytic crisis?
– Anemia + Reticulocytosis = Misleading results
• For females, cytochemical analysis should be
used
• Normal and deficient control blood should
always be used for comparison
– Sheep are naturally deficient, bah!