Chapter 27a

CHAPTER 27
Hemoglobinopathies
Hemoglobinopathy
o disease state (opathy) involving hemoglobin molecule

o most common genetic diseases (affecting 7% of
worlds population)
o results from genetic mutation in one or more genes
that affect hemoglobin synthesis
o mutated genes: code for either proteins that make
up the hgb molecule (globin or polypeptide chains)
or the proteins involved in synthesizing or regulating
synthesis of globin chains
Hemoglobinopathy
o affect hemoglobin synthesis (qualitatively or

quantitatively)
Qualitative
o hemoglobin synthesis occurs at a normal or near-
normal rate
o hgb molecule has an altered amino acid sequence
within the globin chains
structural defect: alters the structure of hgb molecule
qualitative defect: alters its function
Hemoglobinopathy
Quantitative
o thalassemia: reduced rate of hemoglobin
synthesis but do not affect the amino acid
sequence of globin chains
produces anemia and stimulates the production of other
hemoglobins not affected by the mutation in an attempt
to compensate for the anemia
Structure of Globin Genes
o six functional human globin genes located on

two different chromosomes
o , : chromosome 16: -like genes
o ,,,: chromosome 11: -like genes
o one copy of each globin gene per chromatid
o total of two genes per diploid nucleus (exception
of and )
Structure of Globin Genes
o two copies of and genes per chromatid

o total of four genes per diploid nucleus
o each globin gene: codes for
-globin gene (HBA1 and HBA2): template to
synthesize -globin chains
-globin gene (HBB): codes for -globin chain
-globin genes (HBG1 and HBG2): code for -globin
chains
-globin gene (HBD):codes for -globin chain
Hemoglobin Development
o each hgb molecule: four globin chains

pair of -like chains and a pair of -like chains
o embryonic life
activated/ synthesis of production of
first three months 1 -like gene() Hb Gower-1
1 -like gene() ( 2 2 )
shortly thereafter and chains Hb Gower-2
(2 2 )
Hb Portland
(2 2 )
later and chains Hb F or fetal hgb
and ceases (2 2)
Hemoglobin Development
o embryonic life
activated/ synthesis of production of
six months after birth replaced by chain Hb A or adult hgb
chains decreases (2 2 )
around birth chains at low levels Hb A2 or 2nd adult hgb
(2 2 )
o BCL11A and KLF1, zinc-finger transcriptional

repressors: silence the -globin gene and mutations in
the gene that codes for either factor results in elevated
HbF levels
Genetic Mutations
o >1000 structural hgb variants existing

o type of mutations: point mutations, deletions,
insertions, and fusions involving one or more of adult
globin genes- ,,,
Point Mutation
o most common type
o replacement of the original nucleotide in the normal
gene with a different nucleotide
o one nucleotide replaced by another= codon triplet
remains intact, reading frame is unaltered
Genetic Mutations
Point Mutation
o substitution of one amino acid in the globin chain
product at position to the location of the original
point mutation
o 1109 of 1181 know hgb variants: point
mutation=amino acid substitution
o 35: two point mutations in same globin gene= two
amino acid substitution
Genetic Mutations
o Deletion: removal of one or more nucleotides

o Insertions: addition of one or more nucleotides
o Both: not divisible by three and disrupt the reading
frame; 72 cases
leads to nullification of synthesis of the corresponding
globin chain
reading frame remains intact
result: addition or deletion of one or more amino acids
in the globin chain product= affects the structure and
function of the hemoglobin molecule
Genetic Mutations
Chain extensions
o when stop codon is mutated so that translation
continues beyond the typical last codon
o amino acids continue to be added until stop codon
is reached by chance
o globin chains: longer than normal
o globin chain extensions= degradation of globin
chain and quantitative defect
Genetic Mutations
Chain extensions
o if extension of globin chain is insufficient to
produce significant degradation: defect is
qualitative and classified as hemoglobinopathy
o hgb molecules with extended globin chains: fold
inappropriately which affects hgb structure and
function
Genetic Mutations
Gene fusions
o when two normal genes break between
nucleotides, switch positions, and anneal to the
opposite gene
o example: if -globin gene and -globin gene
break in similar locations, switch positions, and
reanneal
resultant genes: and fusion genes= head of the
fusion gene is from one original gene and the tail is from
the other
Genetic Mutations
Gene fusions
o reading frames not disrupted
o similar globin chain lengths
o genes are transcribed and translated into hybrid
globin chains
o fusion chains fold differently
o affect the corresponding hemoglobin function
o 9 identified
Zygosity
o association between the number of gene mutations

and the level of severity of the resultant genetic
defect
o level of severity associated with each gene that is
normally used to synthesize the globin chain
product
o for normal adult globin genes: four copies of
and genes; two copies of and genes
o theory: result in four levels of severity for the
and gene mutations
Zygosity
o if all things were equal: require twice as many

mutations within the and genes to produce the
same physiologic effect as mutations within the
and genes
o and genes are transcribed and translated at
such low levels in adults
mutations of these genes: little impact on overall
hemoglobin function
Zygosity
o Hb A: dominant hemoglobin in adults= composed

of and chains
gene mutations would affect overall hgb function to a
greater extent than the same number of gene
mutations
explains the greater number of identified chain
variants compared with chain variants
single gene mutation: more likely to create a clinical
condition than single gene mutation
Zygosity
o inheritance pattern of chain variants

heterozygous: one gene mutated= trait
homozygous: both genes mutated= disease
Pathophysiology
o manner in which a disorder translates into

clinical symptoms
o impact of point mutations on hemoglobin
function depends on:
chemical nature of the substituted amino acid
where it is located in the globin chain
number of genes mutated (zygosity)
Pathophysiology
o charge and size of substituted amino acid

alter the manner in which globin chain folds
change in charge: affects interaction of
substituted amino acid with adjacent amino acids
change in size: makes the globin chain either
more or less bulky
determine the impact on hgb structure by
potentially altering the tertiary structure of the
globin chain and quaternary structure of the hgb
molecule
Pathophysiology
o changes in hgb structure: affect function

o location of substitution: impact on the degree
of structural alteration and hgb function based
on its positioning within the molecule and
interactions with the surrounding amino acids
o in sickle cell mutation: one amino acid
substitution=hgb polymerization
leading to formation of long hgb crsytals that stretch
the RBC membrane and produce the crescent moon
or sickle cell shape
Pathophysiology
o in -hemoglobinopathies: zygosity predicts two

severities of disease
o homozygous -hemoglobinopathies
both genes mutated
variant hgb becomes the dominant hemoglobin
type
normal hgb (Hb A) is absent
examples: sickle cell disease (SCD, Hb SS) and Hb
C disease (Hb CC)
Pathophysiology
o heterozygous -hemoglobinopathies
one gene mutated and the other is normal
50/50 distribution
variant hgb usually present in lesser amounts than
Hb A: due to an attempt to minimize the impact of
abnormal hgb
in some cases: present in equal amounts
examples: Hb S trait (Hb AS) and Hb C trait (Hb
AC)
Pathophysiology
o homozygous sickle cell disease (Hb SS)

severe form of disease
occurs less frequently
requires lifelong medical intervention (begin early
in life)
o heterozygous sickle cell disease (Hb AS)
more common
rarely experience symptoms
Pathophysiology
o Fishleder and Hoffman: divided structural

hemoglobins into four groups
1. abnormal hgb that results in hemolytic anemia
Hb S and unstable hemoglobins
2. abnormal hgb that results in methemoglobinemia
Hb M
3. hemoglobin with either increased or decreased
oxygen affinity
4. abnormal hgb with no clinical or functional effect
Pathophysiology
o imbalanced chain production: associated with

rare instances with structurally abnormal chain
Hb Lepore
reduced production of the abnormal chain
o many of variants: clinically insignificant= do
not show any physiologic effect
o most clinical abnormalities associated with
chain followed by chain
Pathophysiology
o and chains: small amount involved; rarely

detected and usually no consequence
o Hb S: most frequently occurring of abnormal
hemoglobins and most severe
Nomenclature
o hemoglobins reported in the literature:

designated by letter of the alphabet
o normal adult hgb: Hb A
o fetal hgb: Hb F
o as middle of the alphabet reached: it became
apparent that alphabet would be exhausted
before all mutations were named
o currently: some abnormal hgb assigned a
common designation and scientific designation
Nomenclature
o common name selected by the discoverer and

usually represents the geographic area where
hgb was identified
o single capital letter: indicate a special
characteristic of hgb variants
hgb demonstrating identical electrophoretic mobility
but containing different amino acid substitutions:
Hb G-Philadelphia, Hb G-Copenhagen, and Hb
C-Harlem
Nomenclature
o variant description: also involves scientific

designations that indicate variant chain, the
sequential, and helical number of abnormal
amino acid, and nature of the substitution
o designation [6 (A3) Glu Val] for Hb S
mutation: substitution of valine for glutamic acid
in the A helix in the chain at position 6
Hemoglobin S: Sickle Cell Anemia
History
o origin not identified
o symptoms of disease have been traced in one
Ghanaian family (1670)
o 1910: first report by Chicago cardiologist,
Herrick= West Indian student with severe anemia
o 1917: Emmel recorded that sickling occurred in
nonanemic patients and in patients who were
severely anemic
Hemoglobin S: History
o 1927: Hahn and Gillespie described the pathologic

basis of the disorder and its relationship to the
hemoglobin molecule
sickling occurred when solution of RBCs was deficient
in oxygen
RBC shape was reversible when solution was
oxygenated again
o 1946: Beet reported that malarial parasites were

present less frequently in blood films from patients
with SCD than in individuals without SCD
o sickle cell trait: resistance against infection with
Plasmodium falciparum
occurs early in childhood between the time that passively
acquired immunity dissipates and active immunity
develops
o 1949: Pauling showed that when Hb S is

subjected to electrophoresis, it migrates
differently than does Hb A
caused by an amino acid substitution in the globin
chain
Pauling and coworkers: defined the genetics of the
disorder and distinguished heterozygous sickle trait
(Hb AS) from homozygous state (Hb SS)
o Sickle cell diseases: a group of symptomatic

hemoglobinopathies that have in common sickle cell
formation and associated crises
patients are either homozygous for Hb S (SS)
or compound heterozygotes expressing Hb S in
combination with another hemoglobin chain mutation
(Hb C or -thalassemia)
most common form
Hb SS, Hb SC, Hb S--thalassemia: most frequently
Inheritance Pattern
o hemoglobin variants: autosomal codominants,
with one gene inherited from each parent
o patients with SCD (Hb SS), Hb SC, or Hb S--
thalassemia: inherited a sickle (S) gene from one
parent and an S,C, or -thalassemia gene from the
other
o homozygotes (Hb SS): more severe
o heterozygotes (Hb AS): generally asymptomatic
Prevalence
o highest frequency: Sub-Saharan Africa: 230,000 babies
born each year with SCD (0.74%)
o in five geographic areas
Neonates with Neonates with

Sickle Cell Trait Sickle Cell Disease
Sub-Saharan Africa 64.4% 75.5%
Arab-India 22.7% 16.9%
Americas 7.4% 4.6%
Eurasia 5.4% 3.0%
Southeast Asia 0.1% 0%
Hemoglobin S: Prevalence
o 50% of neonates with SS and AS genotypes:

Nigeria, India, and DR Congo
o in US: SCD found mostly in individuals of African
descent, also in individuals from Middle East,
India, and Mediterranean area
o SCD also found in Carribean and Central and South
America
o sickle cell mutation becoming more prominent in
southern India (part in certain tribes)
Etiology and Pathophysiology

6Glu Val
o structural formula: 22
o on the chain at position 6, glutamic acid is
replaced by valine
o mutation occurs in nucleotide 17, where thymine is
changed to adenine
o resulting in a change in codon 6 and the
substitution of valine for glutamic acid at amino
acid position 6
Hemoglobin S: Etiology
and Pathophysiology
o glutamic acid: net charge of (-1)

o valine: net charge of (0)
o amino acid substitution: change in charge of (+1)
o affects the electrophoretic mobility of hemoglobin
molecule
o also affects the way the hgb molecules interact
with one another within the erythrocyte cytosol
and Pathophysiology
o nonpolar (hydrophobic) valine amino acid: placed in

the position that the polar glutamic acid once held
o polar glutamic acid: chain folds in such a way
that glutamic acid extends outward from the surface
of hgb tetramer to bind water and contribute to hgb
solubility in the cytosol
o hydrophobic valine also extended outward
instead of binding water, it seeks hydrophobic niche with
which to bind
and Pathophysiology
o Hb S fully oxygenated: the quaternary structure of

the molecule does not produce a hydrophobic pocket
for valine to bind to
allows the hgb molecules to remain soluble in the
erythrocyte cytosol like Hb A and maintains the normal
biconcave disc shape of the RBCs
o natural allosteric change change: upon deoxygenation
creates a hydrophobic pocket in the area of phenylalanine
85 and leucine 88
allows valine from an adjacent hgb molecule to bind
and Pathophysiology
o Hgb pairing: creates an orientation that helps other

hemoglobin molecules to form electrostatic bonds
between amino acids and becomes the seed for
polymer formation
o other hgb pairs: polymerize, forming a hgb core
composed of 4 hgb molecules that elongate in a
helical formation
o outer layer of 10 hgb molecules: forms around the
4-hgb-molecule core= long, slender Hb S polymer
and Pathophysiology
o Hb S molecules within the RBCs become less soluble=

forming tactoids or liquid crystals of Hb S polymers
grow in length beyond the diameter of RBC= causing
sickling
o homozygotes: sickling process begins when oxygen
saturation: <85%
o heterozygotes: sickling = <40%
and Pathophysiology
o blood becomes more viscous when polymers are

formed and sickle cells are created
slows blood flow
o decreased oxygen tension and increase in 2,3-BPG
o reduced blood flow: prolongs the exposure of Hb S-
containing erythrocytes to a hypoxic environment
o lower tissue pH: decreases oxygen affinity= further
promotes sickling
and Pathophysiology
o end result: occlusion of capillaries and arterioles by

sickled RBCs and infarction of surrounding tissue
o sickle cells in two forms:
Reversible Sickle Cells
Hb S-containing erythrocytes that change shape in
response to oxygen tension
circulate as normal biconcave discs when fully
oxygenated
and Pathophysiology
Reversible Sickle Cells

undergo hgb polymerization, increased viscosity, and
change shape on deoxygenation
cause casoocclusive complications of SCD
able to travel into the microvasculature in the
biconcave disk conformation due to normal theologic
properties when oxygenated; distorted and viscous as
they deoxygenates
and Pathophysiology
Irreversible Sickle Cells

do not change shape regardless of the change in
oxygen tension or degree of hgb polymerization
seen on peripheral blood film as elongated sickle cells
with a point at each end
recognized as abnormal by the spleen
removed from circulation
prevents them from enterin the microcirculation and
causing vasoocclusion
and Pathophysiology
o level of intracellular hydration: also affects

sickling process
o when RBCs containing Hb S: exposed to low
oxygen tension= hgb polymerization
polymerized deoxyhgb S: activates a membrane
channel (Psickle)= inactive in normal RBCs
membrane channels open when the blood partial
pressure of oxygen decreases to less than 50 mm Hg
and Pathophysiology
o open Psickle channels: allow influx of Calcium

raising the intracellular calcium levels and activating a
second membrane channel (Gardos channel)
o activated Gardos channel: causes efflux of
Potassium
stimulates the efflux of Chloride through another
membrane channel to maintain charge equilibrium
across the RBC membrane
and Pathophysiology
o eflux of ions: leads to water efflux and

intracellular dehydration
effectively increasing intracellular concentration of Hb S
and intensifying polymerization
o K+/Cl- contransporter system: another
contributor to efflux and resultant dehydration
activated by dehydration and positively charged hgb
such as Hb S and Hb C
also activated by low pH in the spleen and kidneys
and Pathophysiology
o oxidative damage triggered by Hb S

polymerization: cause altered function of
membrane channels
o injury to RBC membrane: induces adherence
to endothelial surfaces
causes RBC aggregation, produces ischemia, and
exacerbates Hb S polymerization
and Pathophysiology
o redistribution of phospholipis in the RBC

membrane= hemolysis, vasoocclusive crisis,
stroke, and acute chest syndrome
o in bilayer membranes of normal RBCs:
outer plasma layer: choline phospholipids like
sphingomyelin and phosphatidylcholine
inner cytoplasmic layer: aminophospholipids like
phosphatidylserine (PS) and phosphatidylethanolamine
and Pathophysiology
o asymmetrical distribution of membrane

phospholipids: by adenosine triphosphate-
dependent enzymes (translocases or flippases)
o flippases inhibition and activation of an enzyme
(scramblase): more random distribution of
membrane phospholipids
increases the number of choline phospholipids on the
interior half of the membrane and the number of
aminophospholipids on the exterior membrane surface
and Pathophysiology
o sickle cells of homozygous: express 2.1% PS on

erythrocyte exterior surfaces
0.2% for normal Hb AA controls
o Hb S polymerization: produces microparticles and
iron complexes that adhere to the RBC membrane
generate reactive oxygen species
contribute to flippase inhibition and scramblase activation
along with increased intracellular calcium or protein
kinase C activation
and Pathophysiology
o PS on exterior surface: binds thrombospondin on

vascular endothelial cells
enhance adherance between RBCs and the vessel wall
vasoocclusive crisis, activation of coagulation, and
decreased RBC survival
o RBCs with PS on external surface: vulnerable to
hydrolysis by secretory phospholipase A2 (sPLA2)
results to vascular damage that contributes to acute chest
syndrome
Clinical Features
o from no symptoms to potentially lethal state
o symptoms vary between ethnic groups with Indian
patients expressing a much milder disease than their
African counterparts
o thousand hemoglobin variants known
o only 8 genotypes cause severe disease: Hb SS,
Hb S--thal, severe Hb S-+-thal, Hb SD-Punjab, Hb
SO-Arab, Hb SC-Harlem, Hb CS-Antilles, and Hb S-
Quebec-CHORI
BOX 27-3
Hemoglobin S: Clinical Features
o moderate disease: Hb SC, moderate Hb S-+-thal,

and Hb AS-Oman
o mild disease: mild Hb S-silent -thal, Hb SE, and
Hb SA- Jamaica Plain
o very mild disease: Hb S-HPFH and Hb S with a
variety of mild variants
o symptom variability: due to intracellular ratio of Hb
S to Hb F, as well as factors that affect vessel tone
and cellular activation
o individuals affected: characteristically symptom

free until the second half of the first year of life
due to protective effect of Hb F
o at the end of first 6 months of life: mutated
chains begin to be produced and gradually
replace normal chains
causes increased Hb S and decreased Hb F
o RBCs containing Hb S: susceptible to hemolysis

o progressive hemolytic anemia and splenomegaly
o episode of recurring pain: crises
o sickle cell crises: described by Diggs: any new
syndrome that develops rapidly in patients with
SCD owing to the inherited abnormality
various crises: vasoocclusive or painful, aplastic,
megaloblastic, sequestration, and chronic hemolytic
o hallmark: vasoocclusive crisis (VOC)

acounts for most hospital and emergency
department visits
acute, painful aspect that occurs with great
predictability and severity
triggered by acidosis, hypoxia, dehydration,
infection, fever, and exposure to extreme cold
o Hb S polymerization and sickling of RBCs play a

major role in vasoocclusion in SCD
o most VOC occur in capillaries and postcapillary venules
o list of possible risk factors:
polymerization, decreased deformability, sickle cell-
endothelial cell adherence, endothelial cell activation, WBC
and platelet activation, hemostatic activation, and altered
vascular tone
o during inflammation:
increased WBCs interacting with endothelium
platelet activation= elevation of thrombospondin
clinical dehydration= increase VWF
trigger RBC adherence to endothelium
precipitate vascular obstruction
o mechanism of obstruction: dense cells

less deformable
greatest risk for intracellular polymerization due to highest
Hb S concentration
o vasoocclusive episodes: consume the patient
organ by organ
through destructive and debilitative effects of cumulative
infarcts
o 8-10% of SCD patients: develop cutaneous

manifestations in forms of ulcers or sores on the
lower leg
o abnormal interaction between sickle cells and
vascular endothelium
great impact on the vasoocclusive event
o endothelial adherence: correlates with
severity of painful episodes
o sickle cell adherence to vascular endothelium

results in intimal hyperplasia that can slow blood flow
o Hb SC disease: less sickling with fewer
adherent RBCs
o painful episodes: each persists for 4-5 days
o protracted episodes: last for weeks
o repeated splenic infarcts: produce scarring=
diminished splenic tissue and abnormal function
o splenic sequestration: sudden trapping of

blood in the spleen
rapid decline in hgb: <6 g/dL
occurs most often in infants and young children
(spleens are chronically enlarged)
children: have earlier onset of splenomegaly and
lower level of Hb F at 6 months of age
o crises: associated with respiratory tract infection

o autosplenectomy: gradual loss of splenic function
evidence: presence of Howell-Jolly and Pappenheimer
bodies in RBCs
o pulmonary infarction from sickling in the
microvasculature: causes acute chest syndrome
o Acute Chest Syndrome

fever, chest pain, and presence of pulmonary
infiltrates on the chest radiograph
leading cause of death among adults with SCD
10% of adults with ACS: die from complications
linked to chronic lung disease and pulmonary
hypertension
in children: precipitated by infection; fever, cough,
and tachypnea
o Acute Chest Syndrome

also linked with sPLA2: predictor of ACS in SCD
patients
rises 24-48 hours before symptoms of ACS begin
high sPLA2 level correlates with the degree of
lung damage
o Pulmonary Hypertension (PHT)

serious and potentially fatal sequels of SCD
prevalence: 33%
10% of patients: more severe version
mortality rate: 40% at 40 months

association between development of PHT and
nitrous oxide (NO) pathway
NO produced from the action of endothelial NO
synthase (eNOS) on arginine= vasodilation
SCD patients: decreased NO
leads to vasoconstriction and hypertension
low NO in blood: fail to inhibit endothelin-1
potent vasoconstrictor= additional vasoconstriction
and hypertension

connection between NO and SCD= hemolytic crisis
erythrocyte hemolysis: releases high levels of arginase
degrades arginine
less NO production from eNOS
free hgb released from hemolyzed RBCs:
scavenges NO; reduces the levels and
exacerbates the vasoconstriction and
hypertension

blood arginine and NO levels: drop a few days
before the onset of ACS
finding suggesting that the NO pathway is a
connection between SCD, PHT, and asthma
arginine treatment: reduces pulmonary artery
pressure
effect is not sustainable and does not reduce mortality
o Bacterial infections
major problem for SCD patients
increased susceptibility to life-threatening infection
from S. aureus, S.pneumoniae, and H.influenzae
acute infections: common causes of
hospitalization and most frequent cause of death
o Bacterial infections
Septicemia: bacterial infection of blood
exacerbated by autosplenectomy effect as spleen
gradually loses its ability to function as a
secondary lymphoid tissue to effectively clear
organisms from the blood
o Chronic Hemolytic Anemia

shortened RBC survival of between 16-20 days
decrease in hgb and hct
elevated reticulocyte count and jaundice
continuous screening and removal of sickle cells by
spleen
perpetuate the CHA and autosplenectomy effect

hepatitis and gallstones: also cause jaundice
chronic hemolysis is difficult to diagnose in SC patients
RBC hemolysis: releases free hgb
disrupts the arginine-nitric oxide pathway
results to sequestration and lowering of NO
decreased NO= endothelial cell activation,
vasoconstriction, adherence of RBCs to the endothelium
and pulmonary hypertension

renal dysfunction: another major sequelae
detected early by an increased GFR of 140 mL/min
per 1.73 m found in 71% of SCD patients
progression identified by detecting microalbuminuria
(<4.5 mg/mmol), proteinuria, elevated BUN, and
creatinine levels
angiotensin-converting enzyme inhibitors (ACEI):
lower proteinuria
o Megaloblastic episodes
result from sudden arrest of erythropoiesis due to
folate depletion
folic acid def: cause of exaggerated anemia in
SCD (extremely rare in US)
prophylactic folic acid: prescribed for SCD
patients
o Aplastic episodes (BM failure)

most common life-threatening hematologic
complications
usually associated with infection (parvovirus infection)
clinical problems similar to those seen with other
hemolytic disorders
SC patients: usually can compensate for the decrease
in RBC survival by increasing BM output
o Aplastic episodes (BM failure)

suppressed BM: by bacterial or viral infections
Hct decreases with no reticulocyte compensation
spontaneous recovery phase= presence of nRBCs
and increase in reticulocytes
short-lived and require no therapy
severe anemia: BM remains aplastic, transfusion
necessary
if patients not transfused in a timely fashion= death
o Cardiac defects
enlarged heart and heart murmurs
severe anemia: cardiomegaly develop as heart
works harder to maintain adequate blood flow and
tissue oxygenation
increased cardiac workload with increased BM
erythropoiesis
increases calorie burning= reduced growth rate
at childbearing age: pregnancy becomes risky
o Avascular Necrosis (AVN)

impaired blood supply to head of femur and humerus
in 50% of patients by 35 years of age
physical therapy and surgery
to relieve intramedullary pressure within the bone heads
hip and shoulder implants
necessary in most patients
o Avascular Necrosis (AVN)

leg ulcers: common complication of SCD
ulcers: heal slowly, develop unstable scars, and
recur at the same site
chronic problem with associated chronic pain
o Microstrokes
lead to headaches, poor school performance, reduced
intelligence quotient (IQ), and overt CNS dysfunction
neurologic examination followed by magnetic
resonance imaging (MRI) and transcranial Doppler
ultrasonography or magnetic resonance angiography
recommended to detect microstrokes
Hb S: Incidence with Malaria
and G-6PD Deficiency
o sickle gene: greatest frequency in Central

Africa, the Near East, the region around
Mediterranean, and parts of India
o frequency of gene: parallels the incidence of
P.falciparum
offer protection against cerebral falciparum malaria in
young patients
o malarial parasites: living organisms within

RBCs that use the oxygen within the cells
o reduced oxygen tension= sickling of cells
resulting to injury
injured cells: trapped within the blood vessels of the
spleen and other organs where they are easily
phagocytized by scavenger WBCs
o selective destruction of RBCs containing

parasites:
decreases the number of malarial organisms
increases the time for immunity to develop
o infected cell is uniquely sickled and destroyed
in an area of the spleen or liver
where phagocytic cells are plentiful and oxygen tension is
decreased
o G6PD deficiency: protective effect

o hemolytic episodes: more common in SCD patients
o first 42 months of life
patients with SCD and G6PD deficiency had lower steady-
state hgb levels, higher retic count, 3x more acute
anemia events
more frequent blood transfusions (vasoocclusive and
infectious events than matched sickle cell patient w/o
G6PD def)
o presence of young cells rich in G6PD

increased hemolysis is more likely caused by
enzyme abnormality when the population is shifted
to the oldest cells during an aplastic crisis
Hb S: Laboratory Diagnosis
o anemia of SCD: chronic hemolytic anemia

normocytic, normochromic
Wright-stained blood smear: long, curved cell with a
point at each end= sickle cell
o marked poikilocytosis and anisocytosis with
normal RBCs, sickle cells, target cells, nRBCs
o few spherocytes, basophilic stippling,
Pappenheimer bodies, and Howell-Jolly bodies
o hallmark of SCD: sickle cells and target cells

o moderate to marked polychromasia with
reticulocyte count: 10-25% (hemolytic state and
resultant BM response)
o increased RDW: moderate anisocytosis
o not too elevated MCV: elevated retic ct
o aplastic crisis: decreased retic ct
9
o moderate leukocytosis: 40-50 x10 WBC/ L
o neutrophilia and mild shift toward immature WBCs
o LAP: not elevated when neutrophilia is caused by
sickle cell crisis alone (no underlying infection)
o thrombocytosis
o BM: erythroid hyperplasia= attempt to
compensate for the anemia
o polychromasia and increase retics and nRBCs

o immunoglobulins (particularly IgA): elevated in
all forms of SCD
o normal serum ferritin in young patients but
elevated later in life
o rare hemochromatosis
o chronic hemolysis: elevated levels of indirect
and total bilirubin with jaundice
o two step process

insolubility of deoxygenated Hb S
confirmation of its presence using hemoglobin
electrophoresis, HPLC, or capillary electrophoresis
o more complicated cases: isoelectric focusing,
tandem mass spectrophotometry, or DNA analysis
o older screening test: detects Hb S insolubility

by inducing sickle cell formation on a glass slide
drop of blood mixed with a drop of 2% sodium
metabisulfite (reducing agent) on a slide
mixture is sealed under a coverslip
hgb inside the RBCs is reduced to deoxygenated form
induces polymerization and resultant sickle cell formation
identified microscopically
slow and cumbersome, rarely used
o most common screening test: Hemoglobin

solubility test
decreased solubility of deoxygenated Hb S in
solution= turbidity
blood is added to buffered salt solution with reducing
agent such as sodium hydrosulfite (dithionite) and a
detergent-based lysing agent (saponin)
o Hemoglobin solubility test

saponin: dissolves membrane lipids= release of hgb
from RBCs
dithionate reduces iron from ferrous to ferric
ferric iron: unable to bind oxygen, converting
hemoglobin to deoxygenated form
deoxygenated Hb S: polymerizes in solution= turbid
solutions containing nonsickling hgb= clear

false positive results: hyperlipidemia, few rare
hemoglobinopathies, and when too much blood is
added to the test solution
false negative results: in infants <6 months of age
and with low hematocrits

other positive results: Hb C-Harlem(Georgetown),
Hb C-Ziguinchor, Hb S-Memphis, Hb S-Travis, Hb S-
Antilles, Hb S-Providence, Hb S-Oman, Hb Alexander,
and Hb Porte-Alegre
6Glu Val
have two amino acid substitution ( )
and another unrelated substitution
Hb S-Antilles: cause sickling in heterozygous state
o Alkaline Hgb Electrophoresis

common first step in confirmation of
hemoglobinopathies (SCD)
electrophoresis: based on separation of hgb
molecules in an electric field due primarily to
differences in total molecular charge
AHE: hgb molecules assume a negative charge and
migrate toward the anode (positive pole)

performed on cellulose acetate medium (historically)
replaced by agarose medium
some hgb have same charge and same
electrophoretic mobility patterns
Hgb that exhibit an abnormal electrophoretic pattern at
an alkaline pH may be subjected to electrophoresis at an
acid pH for definitive separation

at acid pH: some hgb assume a negative charge and
migrate toward the anode
other hgb are positively charged and migrate toward the
cathode (negative pole)
Example
Alkaline: Hb S migrates with Hb D and Hb G
: Hb C migrates with Hb E and Hb O
Acid: Hb S separates from Hb D and Hb G
: Hb C separates from Hbe E and Hb O
o HPLC
separated hemoglobin types in a cation
exchange column
requires only one sample injection
identify and quantitate low levels of Hb A2 and Hb F
comigration of Hb A2 and Hb E occurs
o HPLC
best used in diagnosis of thalassemias rather
than hemoglobinopathies
quantitation of low levels of normal and abnormal
hemoglobin levels: to distinguish thalassemia
also used to quantitate Hb A1c levels to monitor
diabetic patients
o Capillary Electrophoresis
separates hemoglobin types based on charge in an
alkaline buffer
use smaller volumes and produces better
separation than traditional agarose electrophoresis
semiautomated systems: Capillary system: allow
for the testing of up to eight samples in parallel
with computerized analysis of results
economical: each capillary can accommodate at
least 3000 runs
o Isoelectric Focusing (IEF)

confirmatory technique
expensive and complex
requires well-trained and experienced laboratoy
personnel
uses an electric current to push the hemoglobin
molecules across a pH gradient
o Isoelectric Focusing (IEF)

charge of the molecules change as they migrate
through the pH gradient until hgb species reaches
its isoelectric point (net charge of zero)
migration stops and hgb molecules accumulate at
their isoelectric position
molecules with isoelectric point difference of as
little as 0.02 pH units: effectively separated
o Neonatal Screening
requires more sophisticated approach
use three techniques: adapted IEF, HPLC, and
reversed-phase HPLC
mutisystem approach that needs to distinguish not
only the multitude of hgb variants but also the
numerous thalassemias
o Hb SS or Hb SC patients: lack normal -globin

chains= no Hb A
o Hb SS: Hb S level: >80%, Hb F: 1-20%
if Hb F >20%: modulate the severity of disease
true in newborns and in patients with hereditary
persistence of fetal hgb
o Hb A2: normal or slightly increased (2-5%)

o Hb A2 quantitation: for differentiating Hb SS
from Hb S--thalassemia
Hb A2: increased
o Hb G, Hb D, and Hb S: all migrate in same
position on alkaline cellulose acetate and alkaline
agarose electrophoresis
Hb G and Hb D: do not give a positive result on the
tube solubility test
o typical sequelae of SCD: may be predicted, and

effectiveness of treatment monitored, if reliable
biomarkers of inflammation can be identified
o common indicators of inflammation
WBC: good predictor of sickle cell events and
mortality
ESR and CRP: exhibit variability too great to
reliably predict events
o common indicators of inflammation

CRP and sPLA2: both elevated during
vasoocclusive crisis and acute chest syndrome
IL-6, IL-10, and protein S: useful indicators
annexin A5: a protein bound to lipids in the
plasma membrane of endothelial cells and platelets
elevate before and during VOC
o lipid damage from oxidative stress: predicted by

plasma elevations of malondialdehyde (MDA) and
depleted -tocopherol
-tocopherol: rises with CRP during bouts of inflammation
o IL-6, IL-10, vascular cell ashesion molecule 1
(VCAM-1) and sPLA2
most promising at predicting impending crisis
Hb S: Treatment
o supportive care: mainstay therapy

o new therapies: modify the genetic pathogenesis of
the disease
o neonatal screening, childhood prophylactic
penicillin therapy, BM transplantation, and
treatment with hydroxyurea (hydroxycarbamide)
in adults: extend life of SCD patients
Hb S: Treatment
o main components of supportive therapy:

adequate hydration, prophylactic vitamin therapy,
avoidance of low-oxygen environments, analgesia for
pain, and aggressive antibiotic therapy with first signs
of infection
o hydration: maintains good blood flow and reduces
vasoocclusive crises
Hb S: Treatment
o prophylactic oral penicillin V: 125 mg dose

twice/day by the age of 3 months-3 years=
recommended to avoid infection, morbidity, and
mortality
o penicillin: increased to 250 mg twice/day from 3-
5 years of age
o during infections: prompt antibiotic treatment
reduces associated morbidity and mortality
Hb S: Treatment
o avoidance of strenous exercise, high altitudes, and

unpressurized air travel: maintains high oxygen
tensions and reduces sickling phenomenon
o painful episodes: treated by ensuring optimal
hydration, rapidly treating associated infection, oxygen
therapy, and effectively relieving pain
o analgesics: foundation of pain management, with
nonsteroidal antiinflammatory drugs (paracetamol and
nefopam)= manage mild ischemic attacks
Hb S: Treatment
o opiods like meperidine or tramadol: for chronic pain

o acute VOC attacks: treated with morphine in the
emergency department or when in transit
o blood exchange transfusion (BET): for severe VOC
attaks and ACS
o painful crises: increase with age, physicians must
be aware of opiate tolerance and rebound pain
following opiate therapy (central sensitization)
Hb S: Treatment
o repeated painful crises: result in hypersensitivity

to repeated pain by:
increasing peripheral inflammation
increased neurotransmitter release
increased calcium influx into postsynaptic junctions and
other pathways that increase pain signals to the brain
o misinterpreted as drug tolerance causing
inappropriate dose escalation or drug-seeking
behavior= inappropriate treatment termination
Hb S: Treatment
o gradual dose reduction: most appropriate

response to opioid tolerance and central
sensitization
to reset pain receptors followed by switching to opioids
that are less sensitive
o patient should be examined on a regular basis
o routine testing done to establish baseline values
for the patient during nonsickling periods
Hb S: Treatment
o hand-foot syndrome: for children younger

than 3 years, characterized by pain and swelling
in the hands and feet
treatment: increasing intake of fluids and analgesics
for pain
o pneumococcal disease: leading cause of
morbidity and mortality in children (younger
than 6 years)
Hb S: Treatment
o heptavalent conjugated pneumococcal vaccine:

recommended at 2,4, and 6 months of age
o 23-valent pneumococcal vaccine: at 2 years
with a booster at age of 5 years
o annual administration of influenza vaccine:
recommended beginning at 6 months of age
o bacterial infection risk: increases in mature
patients with Hb SC disease and homozygous
SCD
Hb S: Treatment
o transfusions: to prevent complications

periodic transfusions: 8 or more/ year; effective
at preventing stroke, symptomatic anemia, brain
injury, priapism, leg ulcers, PHT, and others
to decrease blood viscosity and percentage of
circulating sickle cells in cases of CNS infarction,
hypoxia with infection, stroke, and others
Hb S: Treatment
o Hb SS patients: transfused with normal Hb AA

blood to bring the volume of Hb S: <50%
or to achieve a hgb: 10 g/dL in an effort to prevent
complications in surgery
o maintenance transfusions: given in pregnancy
if mother experiences vasoocclusive or anemia-
related problems or if there are signs of fetal distress
or poor growth
Hb S: Treatment
o transfusion therapy: cause transfusion reactions,

transfusion-related infections, and iron overload
(most frequent)
o iron overload: associated with endocrine
dysfunction and cardiac disease
deferoxamine: effecive in treating iron overload by
chelating and removing excess iron from the body
administered intravenously
Hb S: Treatment
deferasirox: oral iron chelator; consumed in the

morning as a slurry by dissolving several pills
o BM or HSC transplantation
successful for some individuals
few patients qualify due to lack of HLA-matched,
related donors
event free survival rates for patients receiving
transplants from HLA-identical related donors: 80-
90% for SCD
Hb S: Treatment
patients for transplantation: children younger than
age 17 with severe complications (stroke, ACS, and
refractory pain)
morbidity and mortality after transplantation:
increase with age (another restriction)
restores some splenic function
Hb S: Treatment
cord blood stem cells transplantation from HLA-
identical related and unrelated donors: disease-free
survival rate of 90%
primary benefit: banking of cord blood increases
number of units available to achieve an HLA match
utero stem cell transplantation: to produce
engraftment while immune system of the fetus is
prone to HLA tolerance
Hb S: Treatment
o Hydroxycarbamide (hydroxyurea) therapy

relive sickling disorder by increasing the proportion
of Hb F in RBCs of individuals with SCD
given at 25-30 mg/kg
reduce symptoms and prolong life
daily dosing: better Hb F response compared to
sequential weekly dosing
Hb F does not copolymerize with Hb S= production
of Hb F can be sufficiently augmented
Hb S: Treatment

severity of disease expression and number of
irreversible sickle cells: inversely proportional to
the extent to which Hb F synthesis persists
Hb F: 12-20%= little or no anemia; few have
vasoocclusive attacks
:4-5%= modulate disease
:5-12%= suppress severity of hemolysis and
lessen the frequency of severe episodes
Hb S: Treatment

drug compliance: best monitored by increasing MCV
decreasing LD: indicator of treatment response
response to hydroxycarbamide: variable among SCD
patients
high baseline Hb F, neutrophil levels, and retic count:
best predictors of Hb F response
Hb S: Treatment
o prevention of intracellular RBC dehydration

reduces intracellular Hb S polymerization
reducing VOC
o Senicapoc: inhibit Gardos channels and Mg++ to
modulate K+Cl- transport systems
show increased hgb levels and decreased dense RBCs
reduced hemolysis but no clear reduction in VOC
Hb S: Course and Prognosis
o proper management: increased life expentancy

from 14 years (1973) or 50 years (current)
o Hb SC compound heterozygotes average life span
men: 60
women: 68-70
o Hb SS patients: wide range of vocations and
professions
discouraged from jobs that require strenous physical
exertion or exposure to high altitudes or extreme
environmental temperature variations
Hb S: Course and Prognosis
o newborn screening: reduced mortality in

children with SCD by enabling prompt and
comprehensive medical care
o HPLC: most common form of screening
o hemoglobin electrophoresis and genotyping methods:
for confirmation
Hb S: Sickle Cell Trait
o heterozygous state (Hb AS)

o benign condition
o does not affect mortality or morbidity except
under conditions of extreme exertion
o in 8% of African Americans
o also found in Central Americans, Asians, and
people from Mediterranean region
o individuals are asymptomatic

o no significant clinical or hematologic manifestations
o under extremely hypoxic conditions: systemic
sickling and vascular occlusion with pooling of
sickled cells in the spleen, focal necrosis in the
brain, rhabdomyolysis, and even death
o splenic infarcts
in severe respiratory infection, unpressurized flight
at high altitudes, and anesthesia in which pH and
oxygen levels are sufficiently lowered to cause
sickling
o failure to concentrate urine: only consistent
abnormality found in SCT patients
by diminished perfusion of vasa recta of the kidney
impairs concentration of urine by the renal tubules
renal papillary necrosis with hematuria in some px
o connection between strenous exercise and

severe to fatal adverse events
46 cases (39 military recruits and 7 athletes)
causes of deaths: cardiac failures, renal failure,
rhabdomyolysis, and heart illness
opponents of the connection argue that
these events occur in sickle cell-negative people
many people with SCT do not develop adverse events
o strenous exercise & severe to fatal adverse events

opponents of the connection argue that
fatal sickle crisis cannot be adequately established in the
patients encountering events
similar events have not been clearly documented
but military recruits with SCT have 21x greater risk of
exercise-related death than recruits with normal hgb
o Blood film: normal RBC morphology with exception

of few target cells
o No abnormalities in the leukocytes and
thrombocytes
o Hgb solubility screening test: positive
o Diagnosis: presence of Hb S and Hb A on hgb
electrophoresis or HPLC
Electrophoresis
o Hb S: 40% or less
also seen in patients with -thalassemia or iron or
folate deficiency
o Hb A: 60% or more
o Hb A2: normal or slightly increased
o Hb F: within reference interval
no treatment required
life span: not affected
Hemoglobin C
o next hemoglobinopathy after Hb S to be described

o found almost exclusively in African-American
population
o Spaet and Ranney: reported this disease in
homozygous state (Hb CC) in 1953
Hb C: Prevalence, Etiology,
and Pathophysiology
o found in: West African= 17-28%

: African Americans= 2=3%
o most common nonsickling variant encountered in
the United States
o third most common in the world
6Glu Lys
o structural formula: 22
lysine substituted for glutamic acid in position 6 of
the chain
and Pathophysiology
o lysine: +1 charge
o glutamic acid: -1 charge
o result of substitution: net charge of +2
o different structural effect on the hgb molecule
than the Hb S substitution
o inherited in the same manner as Hb S but
manifests milder disease
and Pathophysiology
o polymerizes under low oxygen tension

polymer structure differ with Hb S
Hb S polymers: long and thin
Hb C polymers: short, thick crystal within the RBCs
does not alter RBC shape to the extent that Hb S does
less splenic sequestration and hemolysis
no vasoocclusive crisis
Hb C: Laboratory Diagnosis
o mild to moderate, normochromic, normocytic anemia

o some microcytosis and mild hypochromia
o target cell: marked increase
o reticulocytes: slight to moderate increase
o nRBCs present
o hexagonal crystals of Hb C: form within the
erythrocyte and may be seen on blood film
o extracellular crystals: no evidence of cell membrane

o in some cells: hgb is concentrated within the
boundary of the crystal
o crystals are densely stained and vary in size
appear oblong with pyramid-shaped or pointed ends
seen on wet preparations by washing RBCs and
resuspending them in sodium citrate solution
o hemoglobin solubility test: negative

o definitive diagnosis: electrophoresis or HPLC
o no Hb AA present
o Hb C: >90%
o Hb F: <7%
o Hb A2: approx 2%
o Hb AC trait:
Hb A: 60%
Hb C: 30%
o cellulose acetate electrophoresis at alkaline pH
Hb C migrates in same position as Hb A2, Hb E,
and Hb O-Arab
o citrate agar electrophoresis at acid pH
Hb C separated from other hemoglobins
o no specific treatment
o disorder becomes problematic only if infection
occurs or if mild chronic hemolysis leads to
gallbladder disease
Hb C-Harlem (Georgetown)
o double substitution on chain

o valine substitution for glutamic acid at position 6
of chain (identical to Hb S substitution)
o substitution at position 73 of aspartic acid for
asparagine (same as Hb Korle Bu mutation)
o abnormal hemoglobin migrates with Hb C on
cellulose acetate electrophoresis at alkaline pH
o heterozygotes: asymptomatic
o compound heterozygotes for Hb S and Hb C-
Harlem: crises similar to those in Hb SS disease
o solubility test: positive
o confirmatory: hgb electrophoresis and HPLC
o cellulose acetate at pH 8.4: migrates in C position
o citrate agar at pH 6.2: migration in S position
o few cases identified

o homozygotes: clinical outcomes is uncertain
o heterozygotes: normal
Hb E: Prevalence, Etiology,
and Pathophysiology
o first describe in 1954

o prevalence: 30% in Southeast Asia
o as a result of influx of immigrants from this area,
Hb E prevalence has increased in US
o occurs infrequently in African Americans and
whites
o chain variant: lysine substituted for glutamic
26Glu Lys
acid in position 26 (22 )
and Pathophysiology
o net charge: +2
o no hemoglobin polymerization due to the position
of the substitution
o aa substitution at codon 26: inserts a cryptic
splice site
causes abnormal alternative splicing
decreased transcription of functional mRNA for Hb E
globin chain
and Pathophysiology
o both qualitative defect (due to aa substitution in

globin chain)
o and quantitative defect with -thalassemia
phenotype (due to decreased production of globin
chain)
Hb E: Clinical Features
o homozygous state (Hb EE): mild anemia with

microcytes and target cells
shortened RBC survival time
not associated with clinically observable icterus,
hemolysis, or splenomegaly
main concern: differentiating it from iron
deficiency, -thalassemia trait, and Hb E--thal
o resembles thalassemia trait

o highest incidence: Thailand= where malaria is
most prevalent
P. falciparum multiplies more slowly in Hb EE RBCs
than in Hb AE or Hb AA RBCs
mutation may give some protection against malaria
o Hb E trait: aysmptomatic
o Hb E combined with -thalassemia: more severe
than Hb EE and more closely resembles -
thalassemia major
requires regular blood transfusions
Hb E: Laboratory Diagnosis
o Hemoglobin solubility test: positive

o Confirmatory: electrophoresis or HPLC
homozygotes heterozygotes
Hb E 90% 30-40%
MCV 55-65 fL (low) 65 fL
Target cells few to many present
others normal slight
reticulocyte count erythrocytosis
Hb E: Laboratory Diagnosis
o cellulose acetate electrophoresis at alkaline pH

Hb E migrates with Hb C, Hb O, and Hb A2
o citrate agar electrophoresis at acid pH
Hb E separated from Hb C, but it comigrates with
Hb A and Hb O
Hb E: Treatment and Diagnosis
o no therapy required with the disease and trait

o splenomegaly and fatigue in some patients
o genetic counseling is recommended
o Hb E gene mutations: should be discussed in the
same manner as a mild -thalassemia allele
Hemoglobin O-Arab
o chain variant: lysine substitution for glutamic

acid at amino acid position 121 (22 121Glu Lys )
o rare disorder found in Kenya, Israel, Egypt, and
Bulgaria and in 0.4% of African Americans
o no clinical symptoms except for mild
splenomegaly in homozygotes
Hemoglobin O-Arab
o Hb O-Arab with Hb S: severe clinical conditions

similar to those in Hb SS
o homozygotes: mild hemolytic anemia with many
target cells
o confirmatory: electrophoresis or HPLC
Hemoglobin O-Arab
o cellulose acetate agar at alkaline pH

differentiate Hb O-Arab from Hb-C
o citrate agar at an acid pH
Hb O-Arab: only hemoglobin to move just slightly
away from the point of application toward the
cathode
o no treatment is generally necessary
Hemoglobin D and G
o group of at least 16 chain variants (Hb D) and

6 chain variants (Hb G)
o migrate in alkaline pH at the same position as Hb S
because their and subunits have one fewer
negative charge at an alkaline pH than Hb A
o do not sickle when exposed to reduced oxygen
tension
Hemoglobin D and G
o most variants are named for the place where they

were discovered
o Hb D-Punjab and Hb D-Los Angeles: identical
hemoglobins
Glutamine is substituted for glutamic acid at position
121 in chain (22 121Glu Gln )
o Hb D-Punjab: in 3% of northwestern India
o Hb D-Los Angeles: fewer than 2% of African
Americans
Hemoglobin D and G
o Hb G-Philadelphia
chain variant: substitution of asparagine by lysine at
68 Asn Lys
position 68 (2 2)
most common G variant in African Americans
seen with greater frequency than Hb D variants
also found in Ghana
o do not sickle
Hemoglobin D and G
o alkaline electrophoresis
Hb D and G have same mobility as Hb S
o citrate agar at pH 6.0
Hb D and G separated from Hb S
o these variants should be suspected whenever :
a hgb is encountered that migrates in S position on
alkaline electrophoresis
and if the hgb solubilty test is negative
Hemoglobin D and G
Homozygous (Hb DD)

Hb D 95%
Hb A2 normal amounts
Hb F normal amounts
o Hb DD confused with compound heterozygous

state for Hb D and -thal
differentiated by MCV, Hb A2 levels, and family studies
Hemoglobin D and G
o asymptomatic in heterozygotes state

o Hb D disease (Hb DD): mild hemolytic anemia and
chronic nonprogressive splenomegaly
o no treatment required
Compound Heterozygosity with
Hemoglobin S and Another
-globin Gene Mutation
o inheritance of two different mutant genes that

share a common genetic locus (-globin gene)
o inherited Hb S from one parent and another
chain hemoglobinopathy or thalassemia from
other parent
Compound Heterozygosity with
Hemoglobin S and Another
-globin Gene Mutation
o hemolytic anemia of variable severity

o Hb S with other hemoglobins (Hb E, Hb G-
Philadelphia, and Hb Korle Bu): no clinical
consequence
Hemoglobin SC
o most common compound heterozygous syndrome

o structural defect in hgb molecule in which different
amino acid substitution are found on each of two -
globin chains
o at position 6: glutamic acid is replaced by valine
(Hb S) on one -globin chain and by lysine (Hb C)
on the other -globin chain
o frequency: 25% in West Africa
Hb SC: Clinical Features
o resembles mild SCD

o delayed growth and development in normal
children
o does not produce significant symptoms until
teenage years
o cause all vasoocclusive complications of SCA but
episodes are less frequent and damage is less
disabling
o moderate hemolytic anemia

o moderate splenomegaly
o proliferative retinopathy: more common and
more severe in SCA
o respiratory tract infections with S.pneumonia are
common
o patients live longer than patients with Hb SS

o fewer painful episodes
o associated with considerable morbidity and
mortality
o in US: median life span for men= 60 years
women= 68 years
Hb SC: Laboratory Diagnosis
o normocytic, normochromic anemia

o many features associated with SCA
o hgb: 11-13 g/dL
o reticulocyte count: 3-5%
o few sickle cells, target cells, and intraerythrocytic
crystalline structures
o crystals appear as hybrid of Hb S and Hb C
crystals
o crystals: longer than Hb C crystals but shorter

and thicker than Hb S polymers
often branched
o hemoglobin solubility test: positive
due to presence of Hb S
o cellulose acetate agar
Hb C and Hb S migrate in almost equal amounts
(45%)
normal Hb F
o citrate agar at acid pH (confirmatory)

Hb C separated from Hb E abd Hb O
Hb A2: migrates with Hb C; its quantitation is of no
consequence in Hb SC disease
o Hb A2 determination: vital if patient is suspected
of having Hb C concurrent with -thalassemia
Hb SC: Treatment and Prognosis
o therapy similar to that for SCD

Hb S--thalassemia
o compound heterozygosity for Hb S and -thal

o most common cause of sickle cell syndrome in
patients od Mediterranean descent
o second to Hb SC disease among all compound
heterozygous sickle disorders
o causes a clinical syndrome resembling that of
mild or moderate SCA
Hb S--thalassemia
o severity depends on the chain production of

the affected -thal gene
o if no -globin chain production from -thal
gene: similar clinical course to that of
homozygous SCA
o if there is production of normal -globin chain
(Hb S-+-thal): patients have milder condition
than patients with HB SC
Hb S--thalassemia
o distinguished from SCT individuals because of:

presence of greater amount of Hb S than of Hb A
increased Hb A2 and Hb F
microcytosis from thalassemia
hemolytic anemia
abnormal peripheral blood morphology
splenomegaly
Hb SD and Hb SG-Philadelphia
o Hb SD: compound heterozygous SCA

mild to severe hemolytic anemia because both
chains are affected
some have severe vasoocclusive complications
o Hb SG-Philadelphia: double heterozygous SCA
asymptomatic because Hb G is associated with
gene mutation (sufficient Hb A produced)
o Hb D: in African Americans; usually due to

interaction of Hb S with Hb D-Los Anfeles (Hb D-
Punjab)
o Hb SD: blood film
comparable to those in less severe forms of Hb SS
disease
o cellulose acetate at alkaline pH

Hb D and Hb G comigrate with Hb S
o citrate agar at acid pH
separate Hb S from Hb D and Hb G
o clinical picture: differentiate Hb SD and SG
o Hb SD treatment: similar to that for SCD
patients; according to severity of clinical
condition
Hb S/O- Arab and HbS/D- Punjab
o rare compound heterozygous

o cause severe chronic hemolytic anemia with
vasoocclusive episodes
o both replace glutamic acid at position 121
O-Arab substitutes lysine
D-Punjab substitutes glutamine
o glutamic acid at position 121: located on the

outer surface of hgb tetramer
enhances polymerization process involving Hb S
o cellulose acetate at alkaline pH
Hb S/O-Arab mistaken fro Hb SC
Hb C and Hb O-Arab migrate at same position
differentiation: citrate agar at acid pH
o therapy: similar to that of SCD patients
o alkaline electrophoresis
Hb D-Punjab comigrates with Hb S
mutation look like SCD
o both are not clinically significant in either the
heterozygous or homozygous form
Hb S- Korle Bu
o rare hemoglobin variant

o aspartic acid substitutes for asparagine at
position 73 of the chain
o when inherited with Hb S: interferes with
lateral contact between Hb S fibers by disrupting
the hydrophobic pocket for B6 valine
inhibits Hb S polymerization
o compound heterozygous condition: asymptomatic
Concomitant Cis Mutations
with Hb S
o involves second mutation on the same gene along

with Hb S
Hemoglobin C- Harlem
two substitutions on chain: sickle mutation and
Korle Bu mutation
heterozygotes for Hb C-Harlem: asymptomatic
compound heterozygotes (Hb SHb C-Harlem):
resembles Hb SS clinically
with Hb S
Hemoglobin C- Harlem
hemoglobin solubility test: positive
cellulose acetate at alkaline pH
migrates to the C position
citrate agar at acid pH
migrates to S position
with Hb S
Hb S-Antilles
Hb S mutation with a second substitution of
isoleucine for valine at position 23
Hb S-Oman
Hb S mutation with a second substitution of lysine
for glutamic acid at position 121
second mutation enhances hb S= significant sickling

occur even in heterozygotes
Hemoglobin M
o caused by variety of mutations in -, -, -globin

genes
o result in methemoglobin production
o structural abnormality in the globin portion
o most Hb M involve a substitution of tyrosine for
either the proximal (F8) or the distal (E7)
histidine in , , chains
Hemoglobin M
o substitutions cause heme iron to auto-oxidize=

methemoglobinemia
o iron in the ferric state (Fe3+)
o unable to carry oxygen= cyanosis
o seven hemoglobin variants
chain variants: Hb M-Boston, Hb M-Iwate, Hb
Auckland
chain variants: Hb Chile, Hb M-Saskatoon, Hb M-
Milwaukee-1, and Hb M-Milwaukee-2
Hemoglobin M
o ingestion of oxidant drugs

eg sulfonamides
increase methemoglobin to life-threatening levels
o methemoglobin: causes the blood specimen to
appear brown
causes globin chains to precipitate= Heinz bodies
seen on wet preparations
Hemoglobin M
o Diagnosis: spectral absorption of hemolysate or

by hemoglobin electrophoresis
absorption spectrum peaks: determined at various
wavelengths
unique absorption range of each Hb M variant:
identified when these are compared with the
spectrum of normal blood
Hemoglobin M
o before electrophoresis: all hemoglobin types

are converted to methemoglobin
potassium cyanide is added to the sample
migration differences observed due to an aa
substitution not differences in iron states
on cellulose acetate: Hb M migrates slightly more
slowly than Hb A
electrophoresis performed on agar gel at pH 7.1 for
clear separation
Hemoglobin M
o confirmation: HPLC or DNA-based globin gene

analysis
o no treatment
o diagnosis: to prevent inappropriate treatment
for other conditions (eg cyanotic heart disease)
Unstable Hemoglobin Variants
o result from genetic mutations to globin genes

creating hemoglobin products that precipitate in vivo
o produces Heinz bodies
o causes hemolytic anemia
o more than 140 variants exist
majority are chain variants
most others are chain variants
few and chain variants
o most variants: no clinical significance

o majority has increased oxygen affinity
o 25%:responsible for hemolytic anemia (from
compensated mild anemia to severe hemolytic
episodes)
o congenital nonspherocytic hemolytic anemia or
congenital Heinz body anemia
or unstable hemoglobin disease
o unstable hemoglobin disease

appears at or just after birth depending on globin
chains involved
autosomal dominant
all are heterozygous
homozygous: incompatible with life
o instability of hgb molecule may be due to

substitution of a charged for an uncharged amino
acid in the interior of the molecule
substitution of a polar for a nonpolar amino acid in
the hydrophobic heme pocket
substitution of an amino acid in the and chains at
the intersubunit contact points
replacement of an amino acid with proline in the
helix section of a chain
deletion or elongation of the primary structure
Clinical Features
o detected in early childhood in patients with
hemolytic anemia with jaundice and splenomegaly
o fever or ingestion of an oxidant: worsen hemolysis
o severity of anemia depends on the degree of
instability of the hgb molecule
o unstable hgb: precipitates in vivo and in vitro
in response to factors that do not affect normal hgb (eg
drug ingestion and heat or cold exposure)
Clinical Features
o Heinz bodies: precipitated hgb
attaches to the cell membrane, causing clustering of
band 3, attachment of autologous immunoglobulin, and
macrophage activation
trapped mechanically in the splenic sieve= shortens
RBC survival
abnormal oxygen affinity
Clinical Features
o most prevalent: Hb Koln
o other: Hb Hammersmith, Hb Zurich, Hb Gun Hill
o large variability in the degree of instability
extent of hemolysis varies greatly
o Hb Zurich: presence of an oxidant is required for
any significant hemolysis to occur
Laboratory Diagnosis
o RBC morphology varies
o normal or slight hypochromia
o prominent basophilic stippling
may be caused by excessive clumping of ribosomes
o before splenectomy: hgb= 7-12 g/dL
: retic count= 4-20%
o after splenectomy: anemia is corrected,
reticulocytosis persists
o Heinz bodies in supravital stain
after splenectomy: larger and more numerous
o many patients excrete dark urine with dipyrrole
o many unstable hemoglobin: migrate in the normal
AA patterns
not detected on electrophoresis
o other tests to detect these variants
isopropanol precipitation test
principle: isopropanol solution at 37C weakens
the bonding forces of the hgb molecule
if unstable hgb present: rapid precipitation
occurs in 5 mins, heavy flocculation after 20 mins
normal hgb: precipitate after approx 40 mins
o other tests to detect these variants
Heat Denaturation Test
incubated at 50C for one hour
heat sensitive unstable hgb: flocculent precipitation
normal hgb: little or no precipitation
o significant number of Heinz bodies appear after
splenectomy
o individuals with intact spleen:
with longer incubation and addition of oxidative
substance (eg acetylphenylhydrazine)
unstable hgb: form more Heinz bodies
o isoelectric focusing: resolve many hgb variants
with only a slight alteration in their isoelectric point
o HPLC or DNA-based globin gene analysis
Treatment and Prognosis

o patients treated to prevent hemolytic crises
o severe cases: spleen must be removed to
reduced sequestration and rate of removal of RBCs
o unstable hgb disease is rare= prognosis is unclear
o patients: cautioned against sulfonamide use and
other oxidant drugs
should be informed of the potential for febrile illnesses
to trigger a hemolytic episode
Hgb with Increased and
Decreased Oxygen Affinity
o more than 150 hgb variants

o most are high-affinity variants
o associated with familial erythrocytosis
o others are characterized by low oxygen affinity
o mild to moderate anemia
o normal Hb A: undergoes series of allosteric

conformational changes as it converts from a fully
deoxygenated to fully oxygenated form
conformational changes: affect hgb function and its
oxygen affinity
normal hemoglobin Deoxygenated Oxygenated

(tense) state (relaxed) state
affinity for oxygen and other low high
heme ligands
affinity for allosteric effectors high low
(Bohr protons and 2,3-BPG)
o transition from the tense to relaxed state

involves a series of structural changes that have a
marked effect on hgb function
if aa substitution lowers the stability of the tense
structure: transition occurs at an earlier stage in
ligand binding
hgb has increased oxygen affinity and decreased
heme-heme interaction or cooperativity
Hb Kempsey: aa substitutions at sites crucial to
hgb function
Hemoglobin with
Increased Oxygen Affinity
o high affinity variants: autosomal dominant

o affected individuals: equal volumes of Hb A and
the abnormal variant
exceptions: compound heterozygotes for Hb
Abruzzo and -thal and for Hb Crete and -thal
proportion of abnormal hgb: >85%
Hemoglobin with
o more than 90 variants

o fail to release oxygen on demand= hypoxia
o kidneys: sense the hypoxia;respond by
increasing the release of EPO= compensatory
erythrocytosis
o differ from unstable hemoglobin
abnormal oxygen affinity
do not precipitate in vivo to produce hemolysis
no abnormal RBC morphology
Hemoglobin with
o most are asymptomatic

o no physical symptoms except a ruddy
complexion
o erythrocytosis detected during routine
examinations
high RBC count, hgb, and hct
o normal WBC ct, platelet ct, and peripheral blood
film findings
Hemoglobin with
o hemoglobin electrophoresis: for diagnosis in

some cases
cellulose acetate: abnormal band that separates
from the A band
if no band found: diagnosis of increased oxygen
affinity cannot be ruled out
citrate agar (pH 6.0) or gel electrophoresis: also
used in some cases
Hemoglobin with
o Definitive Diagnosis: oxygen affinity

measurement
o patients live normal lives
o no treatment
o diagnosis made to avoid unnecessary treatment
of the erythrocytosis as a myeloproliferative
neoplasm or secondary erythrocytosis
Hemoglobin with
o quickly release oxygen to the tissues

o normal to decreased hgb concentration
o slight anemia
o Hb Kansas: aa substitution of asparagine by
threonine at position 102 of the chain
o present when cyanosis and normal arterial oxygen
tension coexist
o most may be detected by starch gel
electrophoresis, HPLC, or DNA-based globin gene
analysis
Global Burden of
Hemoglobinopathies
o bulk of the conditions occurs in underdeveloped

countries
o as developing countries work to decrease deaths
from malnutrition, infectious diseases, and other
conditions: more patients will survive and remain
consumers of the health care system
o 1944: thalassemia first identified in Cypress
during the post-World War II recovery period:
decreased dearg rare, increased prevalence
Global Burden of
Hemoglobinopathies
o 1970: in the absence of systems to control the

disease, 78,000 units of blood would be needed
each year
requiring 40% of population to serve as donors
o if left unchecked: cost to maintain thalassemia
therapy would exceed the countrys total health
care budget
o efforts to develop prenatal screening and genetic
counseling programs: reduced SCD birth rate

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CHAPTER 27

o disease state (opathy) involving hemoglobin molecule

o affect hemoglobin synthesis (qualitatively or

o six functional human globin genes located on

o two copies of and genes per chromatid

o each hgb molecule: four globin chains

o BCL11A and KLF1, zinc-finger transcriptional

o >1000 structural hgb variants existing

o Deletion: removal of one or more nucleotides

o association between the number of gene mutations

o if all things were equal: require twice as many

o Hb A: dominant hemoglobin in adults= composed

o inheritance pattern of chain variants

o manner in which a disorder translates into

o charge and size of substituted amino acid

o changes in hgb structure: affect function

o in -hemoglobinopathies: zygosity predicts two

o homozygous sickle cell disease (Hb SS)

o Fishleder and Hoffman: divided structural

o imbalanced chain production: associated with

o and chains: small amount involved; rarely

o hemoglobins reported in the literature:

o common name selected by the discoverer and

o variant description: also involves scientific

o 1927: Hahn and Gillespie described the pathologic

o 1946: Beet reported that malarial parasites were

o 1949: Pauling showed that when Hb S is

o Sickle cell diseases: a group of symptomatic

Neonates with Neonates with

o 50% of neonates with SS and AS genotypes:

Etiology and Pathophysiology

o glutamic acid: net charge of (-1)

o nonpolar (hydrophobic) valine amino acid: placed in

o Hb S fully oxygenated: the quaternary structure of

o Hgb pairing: creates an orientation that helps other

o Hb S molecules within the RBCs become less soluble=

o blood becomes more viscous when polymers are

o end result: occlusion of capillaries and arterioles by

Reversible Sickle Cells

Irreversible Sickle Cells

o level of intracellular hydration: also affects

o open Psickle channels: allow influx of Calcium

o eflux of ions: leads to water efflux and

o oxidative damage triggered by Hb S

o redistribution of phospholipis in the RBC

o asymmetrical distribution of membrane

o sickle cells of homozygous: express 2.1% PS on

o PS on exterior surface: binds thrombospondin on

o moderate disease: Hb SC, moderate Hb S-+-thal,

o individuals affected: characteristically symptom

o RBCs containing Hb S: susceptible to hemolysis

o hallmark: vasoocclusive crisis (VOC)

o Hb S polymerization and sickling of RBCs play a

o mechanism of obstruction: dense cells

o 8-10% of SCD patients: develop cutaneous

o sickle cell adherence to vascular endothelium

o splenic sequestration: sudden trapping of

o crises: associated with respiratory tract infection

o Acute Chest Syndrome

o Acute Chest Syndrome

o Pulmonary Hypertension (PHT)

o Pulmonary Hypertension (PHT)

o Pulmonary Hypertension (PHT)

o Pulmonary Hypertension (PHT)