Hematology 1

Chapter 11- HEMOGLOBINOPATHIES

HEMOGLOBINOPATHIES
E. Elevated HbF in states of erythroid stress and bone marrow
dysplasia
HEMOGLOBINOPATHIES
refers to a disease state involving the Inheritance pattern of β chain variants
hemoglobin molecule. Heterozygous
• All hemoglobinopathies result from a genetic ● When only one β gene is mutated
mutation in one or more genes that affect Homozygous
hemoglobin synthesis. ● When both β genes are mutated
• The hemoglobin molecule has an altered Disease
amino acid sequence within the globin chains ● Homozygous state
which alters the structure of the hemoglobin Trait
molecule (structural defect) and its function ● Heterozygous trait
(qualitative defect).
SICKLES
Point mutation Sickle cell disease
● General term for abnormalities of hemoglobin
– is the most common type of genetic mutation structure.
occurring in hemoglobinopathies. Sickle cell anemia
– is the replacement of one original nucleotide in the ● Most common form of hemoglobinopathy; expression
normal gene with a different nucleotide. of the inheritance of a sickle gene from both parents;
1109 of the 1181 known hemoglobin variants result normocytic normochromic
from a point mutation that causes an amino acid Sickle cell trait (SCT)
substitution. ● is not a disease, but having it means that a person
has inherited the sickle cell gene from one of his or
• On chromosome 16, there are 3 genes within the α her parents.
gene cluster, namely zeta (ζ), alpha 1 (α1), and
alpha 2 (α2). SCDisease-abnormalities
• On chromosome 11, there are 5 genes within the SCAnemia-common form
beta gene cluster, namely epsilon (ε), 2 gamma SCTrait-inherited
genes (γ), a delta gene (δ), and a beta gene (β).
ETIOLOGY
Classification of Hemoglobinopathies ● Inherited from both parents.
● Abnormalities in polymerization with
I.Structural hemoglobinopathies—hemoglobins with deoxygenation=permanent altered protein=sickling
altered amino acid sequences that result in
deranged function or altered physical or chemical
properties
A. Abnormal hemoglobin polymerization—HbS, hemoglobin
sickling
B. Altered O2 affinity
1. High affinity—polycythemia
2. Low affinity—cyanosis, pseudoanemia
C. Hemoglobins that oxidize readily
II. Thalassemias—defective biosynthesis of globin chains
A. α Thalassemias
B. β Thalassemias
C. δβ, γδβ, αβ Thalassemias EPIDEMIOLOGY
III. Thalassemic hemoglobin variants ● Found in African ancestry, Mediterranean, Caribbean,
—structurally abnormal Hb associated with co-inherited South and Central American, Arab and East Indian
thalassemic phenotype ● Confers selective advantage to Plasmodium
A. HbE falciparum malaria
B. Hb Constant Spring ● Sickle cell anemia(the homozygous form of SCD)
C. Hb Lepore ● Most common inherited hematologic disease affecting
humans.
IV. Hereditary persistence of fetal hemoglobin HbS
—persistence of high levels of HbF into adult life ● HbS (α2β2 6 Glu Val) is the result of a single
base-pair change, thymine for adenine, at the sixth
V. Acquired hemoglobinopathies codon of the β globin gene.
A. Methemoglobin due to toxic exposures ● This change encodes valine instead of glutamic acid
B. Sulfhemoglobin due to toxic exposures in the sixth position in the β globin molecule.
C. Carboxyhemoglobin ● It was determined that the sickle cell trait confers a
D. HbH in erythroleukemia resistance against infection with Plasmodium
falciparum.

GARCIA,VERAH EUNICE G. 1
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● Sickle cell anemia, homozygous Hb S, occurs when
both β globin genes have the sickle cell mutation.
● homozygotes (Hb SS) have more severe disease
than individuals who are compound heterozygotes.
● Heterozygotes (Hb AS) are generally asymptomatic.
MEMBRANE ALTERATIONS
● Sickle cells occur in two forms:
1.)Reversible sickle cells are Hb S–containing erythrocytes that
change shape in response to oxygen tension.
2.) irreversible sickle cells do not change their
shape regardless of the change in oxygen tension or degree of
hemoglobin polymerization.
● Erythrocytes containing Hb S become susceptible to
hemolysis, and a progressive hemolytic anemia and
splenomegaly may become evident.
● Splenic sequestration is characterized by a sudden
trapping of blood in the spleen, which leads to a rapid
decline in hemoglobin, often to less than 6 g/dL.
● Gradual loss of splenic function is referred to as
autosplenectomy and is evidenced by the presence of
Howell-Jolly and Pappenheimer bodies in RBCs on
the peripheral blood film.
Clinical features sickle cell anemia SS state
Majority cases present between 3month- 1st year of life .
● Chronic hemolytic anaemia
● Vaso-occlusive events
• Growth and development retarded
• After 6 month- splenomegaly , multiple infarcts,
autosplenectomy
• Recurrent leg ulcers
• Avascular necrosis of femoral head
• Dactylitis (hand-foot syndrome)- first 4 years of life
• Acute infections- pneumonia , meningitis (S.
pneumoniae) , osteomyelitis (salmonella)
(NOTE: NAGIGING SAUSAGE FINGERS!)
• Jaundice , liver enlargment- 1st year of life
• Pigment gall stones
• Acute abdominal pain- visceral infarction
• Priapism
• Acute chest syndrome- fever , chest pain, pulmonary
infarcts.
• Sickle cell retinopathy – ”salmon patches”
intraretinal hemorrhages
Lab Findings
● The peripheral blood film shows marked poikilocytosis
and anisocytosis with normal RBCs, sickle cells,
target cells, nucleated RBCs along with a few
spherocytes, basophilic stippling, Pappenheimer
bodies, and Howell-Jolly bodies.
● immunoglobulin A are elevated in all forms of SCD.
Crises in sickling syndrome
A)Sickling crisis (vaso-occlusive crisis):
• blockage of microcirculation by sickled cells
acute abdominal pain
• Precipated by fever , dehydration , infection.
B) Hemolytic crisis:
NAME
• Further increase in hemolysis with sudden lowering
of Hb, reticulocytosis, jaundice.
C) Aplastic crisis:
• Sudden aplasia of erythroid precursors
• Parvovirus B19
D)Sequestration crisis:
• Massive sequestration of sickled red cells in spleen
and liver with rapid organomegaly
• Reduced blood volume- hypovolemic shock
Acute chest syndrome
● Acute illness with fever and other respiratory
symptoms that displays pulmonary infiltrates on chest
radiographs.
● Third most common death with SCD due to this
complication.
Old method
● A drop of blood is mixed with a drop of 2%
sodium metabisulfite (a reducing agent) on a
slide, and the mixture is sealed under a
coverslip. The hemoglobin inside the RBCs
is reduced to the deoxygenated form; this
induces polymerization and the resultant
sickle cell formation, which can be identified
microscopically.
hemoglobin solubility test
● most common screening test for Hb S
● Blood is added to a buffered salt solution
containing a reducing agent, such as sodium
hydrosulfite (dithionite), and a
detergent-based lysing agent (saponin).
Hemoglobin fractionation and quantification
Alkaline hemoglobin electrophoresis
● Cellulose acetate medium; replaced to agarose
medium
● Common first step in confirmation of
hemoglobinopathies
● Hemoglobin molecules assume a negative charge
and migrate towards the anode
Acid hemoglobin electrophoresis
● Hemoglobin exhibiting an abnormal electrophoretic
pattern at an alkaline pH
● Negative charge migrate towards the anode; positive
charge migrates toward the cathode
● Citrate agar medium
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Isoelectric focusing (IEF) ● Subtype: S β+ thalassemia

● Isoelectric focusing (IEF) is a confirmatory technique - Can make small amount o Hb A and have
that is expensive and complex. less extensive hemolysis and vassoocclusive
● uses an electric current to push the hemoglobin phenomena
molecules across a pH gradient. Hemoglobin S E-thalassemia
TREATMENT ● Hemoglobin E occurs most often among people of
● Prophylactic oral penicillin V at a dose of 125 mg Southeast Asian origin.
twice per day by the age of 3 months to 3 years of ● Most people with sickle-hemoglobin E disease have
age mild to moderate anemia (low blood count).
● The penicillin dosage is increased to 250 mg twice ● Many of the red blood cells in people with
per day from 3 to 5 years of age. sickle-hemoglobin E disease are “stickier” than usual,
● Blood exchange transfusion (BET) is the treatment of and thus may “clog up” the small blood vessels in the
choice for severe VOC attacks and acute chest bones and other parts of the body
syndrome (ACS). ● Painful episodes most often affect the arms, legs,
Sickle Cell Trait stomach, and back.
● refers to the heterozygous state (Hb AS) and Hemoglobin S D-thalassemia
describes a benign condition that generally ● Patients who are homozygous or heterozygous are
does not affect mortality or morbidity except asymptomatic.
under conditions of extreme exertion. ● Some target cells
● The peripheral blood film of a patient with ● Migrates in same position as Hb S and Hb G at an
sickle cell trait shows normal RBC alkaline pH and migrates with Hb A at an acid pH.
morphology, with the exception of a few
Other hemoglobinopathies
target cells.
• Hb D Punjab – at 121 position of beta globin chain glutamic
Diagnosis acid->glutamine
- Electrophoretic pattern containing both Hb A and Hb • Hb D trait (Hb AD)
S, with more Hb A than Hb S. • HbD disease (HbDD)
- The hemoglobin solubility screening test yields • Hb D beta thalassemia
positive results, and sickle cell trait is diagnosed by • HbD Iran
detecting the presence of Hb S and Hb A on • HbD lepre disease
hemoglobin electrophoresis or HPLC. • Hb M
- No treatment is required for this benign condition, and • Hb Saskatoon
the patient’s life span is not affected by sickle cell trait. • Hb C trait
• Hb E disorder
• Hb C disease
Hb E Disorders
Substitution of glutamic acid by lysine on 26 position of beta
globin chain forms Hb E
● On electrophoresis HbE is slow moving ,
migrates in same position as HbC and Hb A2
Hb E trait:
● Clinically normal
● PBF- Microcytosis (MCV 60-80 fl)
Hb E Disease: HbEE
● Mild anemia and splenomegaly, mild
jaundice
● PBF – marked anisocytosis and microcytosis
, target cells , hypochromia.
HEMOGLOBIN C
● It is the most common non sickling variant
encountered in the United States and the third most
common in the world.
● the structural formula a2b2 6Glu Lys, in which lysine
is substituted for glutamic acid in position 6 of the b
Other Sickling syndromes chain.
Laboratory findings
● There is a marked increase in the number of
Hb S beta thalassemia
target cells, a slight to moderate increase in
● Other name: Sickle β-thalassemia
the number of reticulocytes, and nucleated
● Inheritance of the sickle gene from one
RBCs may be present in the peripheral
parent and a β-thalassemia gene from the
blood.
others results in the compound heterozygous
● Hexagonal crystals of Hb C form within the
state
erythrocyte and may be seen on the
● Moderate severe hemolytic anemia
peripheral blood film.
● Splenomegaly in 70% cases
● Hb C yields a negative result on the
● Subtype: S β0-thalassemia
hemoglobin solubility test, and definitive
● Patients who are unable to produce any Hb
A

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diagnosis is made using electrophoresis or hydrophobic pocket for β6 valine, which inhibits Hb S
HPLC. polymerization.
HEMOGLOBIN C-HARLEM (HEMOGLOBIN
C-GEORGETOWN) Hemoglobin M
Has two substitutions on the β chain: sickle mutation and ● Caused by mutations in α-, β- ,and γ-globin genes, all
Korle-Bu mutation of which result in production of methemoglobin.
● Heterozygous Hb C- Harlem: asymptomatic ● Has iron in ferric state and is unable to carry oxygen
● Compound heterozygous Hb SC-Harlem resembles producing cyanosis.
Hb SS clinically ● Hb M variants have altered oxygen affinity and are
● A positive solubility test result may occur with Hb inherited as autosomal dominant disorders.
C-Harlem, and hemoglobin electrophoresis or HPLC ● Invididuals have 30-50% methemoglobin and may
is necessary to confirm the diagnosis. appear cyanotic.
HEMOGLOBIN E ● Diagnosis is made by spectral absorption of
● The variant has a prevalence of 30% in Southeast hemolysate or by hemoglobin electrophoresis
Asia. Hgb variants affecting α or β chains:
● is a b chain variant in which lysine is substituted for ● Hb M-Boston (α chains)
glutamic acid in position 26 (a2b26Glu Lys). ● Hb M-Iwate(α chains)
● both a qualitative defect (due to the amino acid ● Hb Auckland(α chains)
substitution in the globin chain) and a quantitative ● Hb Chile (β chains)
defect with a b-thalassemia phenotype (due to the ● Hb M- Saskatoon(β chains)
decreased production of the globin chain). ● Hb M-Milwaukee-1 (β chains)
Laboratory findings ● Hb M-Milwaukee-2(β chains)
● The homozygous state (Hb EE) manifests as a mild Variants affecting γ chains:
anemia with microcytes and target cells. ● Hb F-M-Osaka
● The main concern in identifying homozygous Hb E is ● Hb F-M-Fort Ripley
differentiating it from iron deficiency, b-thalassemia -Symptoms disappear when Hb A replaces Hb F at age 3 to 6
trait, and Hb E–b-thal. months.
● does not produce a positive hemoglobin solubility test
result and must be confirmed using electrophoresis or
HPLC. “Kung kaya ng iba, kaya mo din…for your future mo yan”
● No therapy is required with Hb E disease and trait.
HEMOGLOBIN O-ARAB
● b chain variant caused by the substitution of lysine for
glutamic acid at amino acid position 121 (a2b2
121Glu Lys).
● Hb O-Arab is the only hemoglobin to move just
slightly away from the point of application toward the
cathode on citrate agar at an acid pH.
● Homozygous individuals have a mild hemolytic
anemia, with many target cells on the peripheral blood
film and a negative result on the hemoglobin solubility
test.
HEMOGLOBIN D AND HEMOGLOBIN G
● Hb D-Punjab and Hb D-Los Angeles are identical
hemoglobins in which glutamine is substituted for
glutamic acid at position 121 in the b chain (a2b2
121Glu Gln).
● Hb G-Philadelphia is a chain variant of the G
hemoglobins, with a substitution of asparagine by
lysine at position 68(a2b2 68Asn Lys).
● Hb D and Hb G do not sickle and yield a negative
hemoglobin solubility test result.
● Hb SC is the most common compound heterozygous
syndrome that results in a structural defect in the
hemoglobin molecule in which different amino acid
substitutions are found on each of two b-globin
chains.
● Hb SC crystals often appear as a hybrid of Hb S and
Hb C crystals.
Hemoglobin S-Korle Bu
● Rare hgb variant with a substitution of aspartic acid
for asparagine at position 73 of the β chain.
● When inherited with Hb S, it interferes with lateral
contact between Hb S fibers by disrupting the

NAME 4
Garcia,Verah Eunice G. BSMT 3
HEMATOLOGY 1
Chapter 12
THALASSEMIA
THALASSEMIA
● Thalassemia is an inherited blood related disorder
due to absent or reduced production of hemoglobin, a
protein present in red blood cells responsible for
carrying oxygen through the body.
● A hemoglobin molecule has two sub-units commonly
referred to as alpha and beta. Both sub-units are
necessary to bind oxygen and deliver it to cells and
tissues in the body. A lack of a particular subunit
determines the type of the resulting thalassemia
(alpha or beta).
● Thalassemia is derived from the Greek word
“thalassa” meaning “the sea” because the
condition was first described in populations living near
the Mediterranean Sea.
● It is now very well known that alpha- and
beta-thalassemia are the most common inherited
single-gene disorders.
RISK FACTORS
● Family history :
Thalassemias are inherited— that is, they're passed from
parents to children through the genes. If person’s parents have
missing or altered hemoglobin-making genes, person may
have thalassemia.
● Ancestry :
Alpha thalassemias most often affect people of Southeast
Asian, Indian, Chinese, or Filipino origin or ancestry.
Beta thalassemias most often affect people of Mediterranean
(Greek, Italian , & Middle Eastern), Asian, or African origin or
ancestry.
GENETIC DEFECTS CAUSING THALASSEMIA
• Reduced or absent transcription of messenger ribonucleic
acid(mRNA) due to mutations in the promoter region or
initiation codon of a globin gene, as well as mutations in
polyadenylation sites that decrease mRNA stability.
• mRNA processing errors due to mutations that add or remove
splice sites resulting in no globin chain or altered globin chain
production
• Translation errors due to mutations that change the codon
reading frame (frameshift mutations), substitute an incorrect
amino acid codon (missense mutations), add a stop codon
causing premature chain termination (nonsense mutations), or
remove a stop codon, which results in an elongated
Deletion of one or more globin genes resulting in the lack of
production of the corresponding globin chains.
TYPES OF THALASSEMIA
ALPHA THALASSEMIA
● Alpha thalassemia is the result of changes in the
genes for the alpha globin component in hemoglobin.
● Alpha globin genes on chromosome 16.
● There is a need for four genes (two from each
parent) to make enough alpha globin protein chains. If
one or more of the genes is missing, people have
alpha thalassemia trait or disease. This means that
the body doesn't make enough alpha globin protein.
● The notation for the normal a2- globin gene complex
or haplotype is αα, which signifies the two normal
genes (α2 and α1 ) on chromosome 16.
● A normal genotype is αα/αα.
TWO HAPLOTYPES OF Α- THALASSEMIA
● α0- thalassemia (α-thal-1)
❖ Deletion of both α chain production from the
chromosome
❖ Designation: - -
❖ α0 haplotype is found in 4% of the population
in Southeast Asia
● α+ -thalassemia
❖ Deletion or non-deletional mutation in one of
the two α – globin gene on chromosome 16
resulting to decreased α chain production.
❖ Designation: - α
❖ Most common of the α-thalassemia
haplotypes
CLINICAL SYNDROMES OF Α-THALASSEMIA
● If person only missing one gene, that person
is a Silent Carrier. This means you won't
have any signs of illness.
● If person missing two genes, person have
Alpha Thalassemia Minor. Person may
have mild anemia.
● If person missing three genes, person likely
will have Hemoglobin H disease (which a
Blood test can detect). This form of
Thalassemia causes moderate to severe
anemia.
● Very rarely, a baby will be missing all four
genes. This condition is called Alpha
Thalassemia Major or Hydrops fetalis.
Babies who have hydrops fetalis usually die
before or shortly after birth.
SILENT CARRIER STATE
● The deletion of one a-globin gene, leaving
three functional a-globin genes (– a/aa), is
the major cause of the silent carrier state.
● Because one a-globin gene is absent, there
is a slight decrease in a chain production.
 A-THALASSEMIA MINOR
● Deletion of two a-globin genes is the major
cause of a-thalassemia minor.
● It exists in two forms: homozygous a1 (– a/–
a) or heterozygous a0 (– –/aa).
● This syndrome is asymptomatic and
characterized by a mild anemia (typical
hemoglobin concentration is 12 to 13 g/dL)
with microcytic, hypochromic RBCs.
HEMOGLOBIN H DISEASE
● Deletion of three a-globin genes is the major cause of
Hb H disease in which only one a-globin gene
remains to produce a chains (– –/– a).
● It is characterized by the accumulation of excess
unpaired b chains that form tetramers of Hb H in
adults.
● characterized by a mild to moderate, chronic
hemolytic anemia with hemoglobin concentrations
averaging 7 to 10 g/dL, and reticulocyte counts of 5%
to 10%.
HB BART HYDROPS FETALIS SYNDROME
● Homozygous a0-thalassemia (– –/– –)
results in the absence of all a chain
production and usually results in death in
utero or shortly after birth.
● The fetus is delivered prematurely and is
usually stillborn or dies shortly after birth.
● anemia, edema, and ascites, the fetus has
gross
● hepatosplenomegaly and cardiomegaly.
DE NOVO AND ACQUIRED Α-THALASSEMIA
● α-Thalassemia with mental retardation
syndrome (ATR):
● Due to large deletions on chromosome 16
involving the α-globin genes
● Due to mutations of the ATRX transcription
factor gene on chromosome X
● α-Thalassemia associated with
myelodysplastic syndromes (ATMDS):
● Due to mutations of the ATRX gene
BETA THALASSEMIA
● Person need two genes (one from each parent) to
make enough beta globin protein chains. If one or
both of these genes are altered , person will have
beta thalassemia. This means that person body
doesn’t make enough beta protein.
● If person have one altered gene , he/she is a carrier.
This condition is called beta thalassemia trait or beta
thalassemia minor. It causes mild anemia.
● If both genes are altered , person will have beta
thalassemia intermedia or beta thalassemia major
(also called Cooley’s anemia). The intermedia form of
the disorder causes moderate anemia. The major
form causes severe anemia.
SILENT CARRIER STATE
● The designation b silent includes the various
heterogeneous b-globin gene mutations that produce
only a small decrease in production of the b chains.
● The silent carrier state (b silent/b) results in nearly
normal a-b chain ratios and no hematologic
abnormalities.
B-THALASSEMIA MINOR
● Results when one b-globin gene is affected by a
mutation that decreases or abolishes its expression,
whereas the other b-globin gene is normal
(heterozygous state).
● reticulocyte count is within the reference interval or
slightly increased.
● Some degree of poikilocytosis (including target cells
and elliptocytes) and basophilic stippling in the RBCs
may be seen on a Wright-stained peripheral blood
film.
B-THALASSEMIA MAJOR
● characterized by a severe anemia that requires
regular transfusion therapy.
● It is usually diagnosed between 6 months and 2 years
of age (after completion of the g to b switch) when the
child’s Hb A level does not increase as expected.
● marked microcytosis, hypochromia, anisocytosis, and
poikilocytosis, including target cells, teardrop cells,
and elliptocytes.
● RBC inclusions are commonly found, including
basophilic stippling, Howell-Jolly bodies, and
Pappenheimer bodies.
B-THALASSEMIA INTERMEDIA
● Thalassemia intermedia is a term used to describe
anemia that is more severe than b-thalassemia minor
but does not require regular transfusions to maintain
hemoglobin level and quality of life
(transfusion-independent).
OTHER THALASSEMIAS CAUSED BY DEFECTS
IN THE B-GLOBIN GENE CLUSTER
THALASSEMIAS WITH INCREASED LEVELS OF FETAL
HEMOGLOBIN
● hereditary persistence of fetal
hemoglobin (HPFH)
➔ the b-globin gene cluster typically contains a deletion
in the db region that leads to the increased production
of Hb F. However, there are also HPFH conditions
that have intact b-globin gene clusters with
non-deletional mutations in the promoter region of the
g-globin genes that lead to the increased Hb F
production.
● δβ°-thalassemia
➔ the increase in production of the g chains is not
sufficient to completely restore the balance between
the a and non-a chains.
● Hemoglobin Lepore Thalassemia
➔ structural variant and rare type of db-thalassemia
caused by a fusion of the db-globin genes.
➔ This mutation occurs during meiosis due to
nonhomologous crossover between the d-globin locus
on one chromosome and the b-globin locus on the
other chromosome.
THALASSEMIA ASSOCIATED WITH
STRUCTURAL HEMOGLOBIN VARIANTS
Hemoglobin S-Thalassemia
● Sickle cell anemia (Hb SS)- a-thalassemia is a
genetic abnormality due to the coinheritance of two
abnormal b-globin genes for Hb S and an
a-thalassemia haplotype.
Hemoglobin C-Thalassemia
● -Hb C-b-thalassemia produces moderately severe
hemolysis, splenomegaly, hypochromia, microcytosis,
and numerous target cells.
Hemoglobin E-Thalassemia
● Hb E-b-thalassemia is a significant concern in
Southeast Asia and Eastern India owing to the high
prevalence of both genetic mutations.
● Hb E is due to a point mutation that inserts a splice
site in the b-globin gene, and results in decreased
production of Hb E.
SIGNS AND SYMPTOMS
● Anemia
● Dark urine (a sign that red blood cells are breaking
down)
● Yellow or Pale skin
● Delayed growth and development
● Weakness
● Fatigue
● Hepatomegaly
● Bone defects (especially bones in the face)
COMPLICATION
● Hepatic Failure
● Congestive Cardiac Failure
● Gall stone
● Hemochromatosis
● Transfusion related Infection
● Hypothyroidism
● Hypogonadism
● Skeletal complication and Multi-Organ Dysfunction
DIAGNOSTIC EVALUATION
● History of patient (Symptoms of Anemia ,Positive
family History , FTT)
● Physical Examination (Severe Anemia , Thalassemic
faces , Hepatosplenomegaly , Growth retardation)
● Lab Investigation (CBC , Hemoglobin Estimation ,
Serum Bilirubin , Peripheral Smear, liver function test)
● Screening Family Members
MEDICAL MANAGEMENT
BLOOD TRANSFUSION
-Chidren who can not maintain haemoglobin level about 7
gm/dl.
-To prevent chronic hypoxia and to supress ineffective
erythropoiesis.
● Transfusions of red blood cells are the main treatment
for people who have moderate or severe
thalassemias. This treatment gives person healthy red
blood cells with normal hemoglobin.
● The procedure usually takes 1 to 4 hours.
● Red blood cells live only for about 120 days. So, may
need repeated transfusions to maintain a supply of
healthy red blood cells.
● If person have hemoglobin H disease or beta
thalassemia intermedia, person may need blood
transfusions on occasion. For example, person may
need this treatment when person have an infection or
other illness, or when anemia is severe enough to
cause tiredness.
● If person have beta thalassemia major, or Cooley's
anemia, person need regular blood transfusions
(often every 2 to 4 weeks). These transfusions will
help maintain normal hemoglobin and red blood cell
levels.
● Blood transfusions allow person to feel better, enjoy
normal activities, and live into adulthood. This
treatment is lifesaving, but it's expensive and carries a
risk of transmitting infections and viruses (for
example, hepatitis). However, the risk is very low in
the United States because of careful blood screening.
IRON CHELATION THERAPY
● Hemoglobin in red blood cells is an iron-rich protein,
regular blood transfusions can lead to a buildup of
iron in the blood. This condition is called iron
overload. It damages the liver, heart, and other parts
of the body.
● To prevent this damage, iron chelation therapy is
needed to remove excess iron from the body. Two
medicines are used for iron chelation therapy
● Deferoxamine is a liquid medicine that's given slowly
under the skin, usually with a small portable pump
used overnight. This therapy takes time and can be
mildly painful. Side effects include problems with
vision and hearing.
● Deferasirox is a pill taken once daily. Side effects
include headache, nausea (feeling sick to the
stomach), vomiting, diarrhea, joint pain, and fatigue
(tiredness).
FOLIC ACID SUPPLEMENTATION
● Folic acid is a B vitamin that helps build healthy red
blood cells. person may need to take folic acid
supplements in addition to treatment with blood
transfusions and/or iron chelation therapy.
● Folic acid supplementation are recommended as iron
therapy & dietary iron should be avoided to prevent
more iron deposition
BONE MARROW TRANSPLANTATION
● A blood and marrow stem cell transplant replaces
faulty stem cells with healthy ones from another
person (a donor). Stem cells are the cells inside bone
marrow that make red blood cells and other types of
blood cells.
● A stem cell transplant is the only treatment that can
cure thalassemia. But only a small number of people
who have severe thalassemias are able to find a good
donor match and have the risky procedure.
● Defective stem cells are replaced by normal stem
cells.
● It is Extremely Expensive and possible in only very
selected cases.
GENE THERAPY
● In gene therapy insertion of normal gene is done in
the stem cells to correct underlying defect .
● In gene manipulation excess of alpha chains is
decreased by increasing the gamma chains .
 ● SA, sickle cell trait
● SS, sickle cell anemia/disease
DIET AND SUPPLEMENTS ● SC, HbSC disease
● Oral folate at minimum 1 mg daily. ● S/β thal, sickle β-thalassemia disease
● Avoid iron rich food such as red meat and ● S with other Hb variants: D, O-Arab
iron fortified cereals or milk. B. Hemoglobins with decreased stability (unstable
● Tea may help decrease intestinal iron hemoglobin variants)
absorption. ● Mutants causing congenital Heinz body
● Dairy products are recommended as they hemolytic anemia
are rich in calcium. ● Acquired instability—oxidant hemolysis:
Drug induced, G6PD deficiency
C. Hemoglobins with altered oxygen affinity
GET ONGOING MEDICAL CARE ● High oxygen affinity states:
Keep your scheduled medical appointments and get any ● Fetal red cells
tests that your doctor recommends. These tests may ● Decreased 2,3-BPG
include: ● Carboxyhemoglobinemia, HbCO
● Low oxygen affinity states:
Monthly complete blood counts and tests for blood
● Increased RBC 2,3-BPG
iron levels every 3 months
D. Methemoglobinemia
Yearly tests for heart function, liver function, and viral ● Congenital methemoglobinemia
infection (for example, hepatitis B and C and HIV) ● Cytochrome b5 reductase deficiency
Yearly tests to check for iron buildup in your liver ● Acquired (toxic) methemoglobinemia
Yearly vision and hearing tests E. Posttranslational modifications
● Nonenzymatic glycosylation
Regular checkups to make sure blood transfusions ● Amino-terminal acetylation
are working ● Amino-terminal carbamylation
SURGICAL MANAGEMENT ● Deamidation

● Splenectomy : It leading to excessive destruction of Diagnosis of thalassemia

erythrocytes and increasing need for frequent blood CBC with PBS
transfusion which result in further iron accumulation. ● Hgb and hct are decreased
● RBC indices decreased
● Cholecystectomy : patients with thalassemia minor ● RDW elevated
may have bilirubin stones in their gallbladder and , if ● Target cell, elliptocytes, polychromasia, basophilic
symptomatic , may require treatment. Perform a stippling, Howell-Jolly bodies, Pappenheimer bodies,
cholecystectomy using a laproscopic or carry out the and nRBCs.
procedure at the same time as the splenectomy Reticulocyte count
● Elevated
Supravital staining
SUMMARY
● Brilliant cresyl blue or new methylene blue
● Classification of hemoglobinopathy Alkali denaturation test
• QUANTITATIVE DISORDERS OF GLOBIN CHAIN ● Quantifying hb F
SYNTHESIS/ACCUMULATION Kleihauer-Betke test
The thalassemia syndromes ● For estimation of fetal-maternal hemorrhage
A. ) β-Thalassemia
● β-Thalassemia minor or trait
● β-Thalassemia major
● β-Thalassemia intermedia
● Lepore fusion gene
● δβ-Thalassemia
● β-Thalassemia with other variants:
● HbS/β-thalassemia
● HbE/β-thalassemia
B.) α-Thalassemia
● Deletions of α-globin genes:
● One gene: α+-thalassemia
● Two genes in cis: α0-thalassemia
● Three genes: HbH disease
● Four genes: Hydrops fetalis
● De novo and acquired α-thalassemia
● α-Thalassemia with mental retardation
syndrome (ATR)
● α-Thalassemia associated with
myelodysplastic syndromes (ATMDS)

● II. QUALITATIVE DISORDERS OF GLOBIN

STRUCTURE
A. Sickle cell disorders
Garcia,Verah Eunice G. BSMT 3
HEMATOLOGY 1
Chapter 14 - ESR and HEMATOCRIT
ESR AND HEMATOCRIT
HEMATOCRIT
● It is the volume of packed red blood cells that occupies a
given volume of whole blood.
● This is often referred to as the packed cell volume (PCV). It
is reported either as a percentage (e.g., 36%) or in liters per
liter (0.36 L/L).
● The values of the duplicate hematocrits should agree within
1%(0.01 L/L)
Why would you get a hematocrit test?
A hematocrit test can help your doctor diagnose you with a particular
condition, or it can help them determine how well your body is
responding to a certain treatment. The test can be ordered for a variety
of reasons, but it’s
most often used to test for:
1. anemia
2. leukemia
3. dehydration
4. dietary deficiencies
What if my hematocrit levels are too low?
Low hematocrit levels may be a sign of:
1. bone marrow diseases
2. chronic inflammatory disease
3. deficiencies in nutrients such as iron, folate, or vitamin B-12
4. internal bleeding
5. hemolytic anemia
6. kidney failure
7. leukemia
8. lymphoma
9. sickle cell anemia
RULE OF THREE
● Used to check results of hemoglobin and
hematocrit
● Applies to samples that have normocytic
normochromic RBCs
● The value of hematocrit must be three times the
value of hemoglobin plus or minus 3.
● Hgb x 3 = hct +/- 3
SOURCES OF ERROR AND COMMENTS
● Improper sealing of the capillary tube causes a
decrease hematocrit reading as a result of leakage
of blood during centrifugation.
● An increased concentration of anticoagulant (short
draw in an evacuated tube) decreases the
hematocrit reading as a result of red blood cell
shrinkage.
● A decreased or increased result may occur if the
specimen was not mixed properly.
● Insufficient centrifugation or a delay in reading
results after centrifugation causes hematocrit
readings to increase.
● The buffy coat of the sample should not be
included in the hematocrit reading because this
falsely elevates the result.
● A decrease or increase in the readings may be
seen if the microhematocrit reader is not used
properly.
● Many disorders, such as sickle cell anemia,
macrocytic anemias, hypochromic anemias,
spherocytosis, and thalassemia, may cause
plasma to be trapped in the red blood cell layer
even if the procedure is performed properly.
● A temporarily low hematocrit reading may result
immediately after a blood loss because plasma is
replaced faster than are the red blood cells.
● The fluid loss associated with dehydration causes
a decrease in plasma volume and falsely
increases the hematocrit reading.
● The introduction of interstitial fluid from a skin
puncture or the improper flushing of an
intravenous catheter causes decreased hematocrit
readings.
FALSE DECREASE
● Low total protein
● Presence of cold agglutinins
FALSE DECREASE
● Increased WBC count
OTHER INSTRUMENTS USED IN MEASUREMENT OF HEMATOCRIT
● ABL 77
● IRMA
● Gem Premier
ERYTHROCYTE SEDIMENTATION RATE
● For detection or monitoring of inflammatory
conditions.
● The ESR is the distance in millimeters that the red
blood cells fall in 1 hour.
● Normal red blood cells have a relatively small
mass and settle slowly.
● The ESR is directly proportional to the red blood
cell mass and inversely proportional to plasma
viscosity.
● The ESR is slower to respond to changes in
● acute disease activity and is insensitive to small
changes in disease activity.
● The conventional manual ESR method is simple,
cheap and independent of a power
● supply, thus making it suitable for a screening
● test.
THREE STAGES OF ERYTHROCYTE SEDIMENTATION
• Stage 1: Formation of rouleaux or lag phase (10 minutes): Red cells
stack together like a pack of coins.
● The ESR depends mainly on this stage.
• Stage 2: Sinking of rouleaux or decantation phase (40 minutes):
● Rapid and constant sedimentation.
● The longer the tube, the longer is this stage and higher the
value of ESR.
• Stage 3: Packing of rouleaux (10 minutes):
● Slow sedimentation.
MODIFIED WESTERGREN ERYTHROCYTE SEDIMENTATION RATE
● The most commonly used method today is the
modified Westergren method.
● One advantage of this method is that the
● taller column height allows the detection of highly
elevated ESRs.
WINTHROBE ERYTHROCYTE SEDIMENTATION RATE
● When the Winthrobe method was first introduced, the
specimen used was oxalate-anticoagulated whole blood and
was placed in a 100-mm column.
 ● Today, EDTA-treated or citrated whole blood is used with the
shorter column. The shorter column height allows a
somewhat increased sensitivity in detecting mildly elevated
ESRs.
● In normal blood, the RBCs remain more or less separated.
They are negatively charged and, therefore repel each other.
( Zeta Potential)
● Increased plasma protein concentration, most notable
fibrinogen, immunoglobulins and acute phase proteins (C SEDIMENT
reactive protein,ceruloplasmin) cause a reduction in the
negative charge of the RBCs and facilitates formation of
rouleaux.

FACTORS AFFECTING ERYTHROCYTE

SEDIMENTATION RATE
● Factors increasing ESR
– Old age
– Pregnancy
– Anemia
– Elevated fibrinogen ROLLER 20LC
– Macrocytosis ● Roller 20 LC instrument can measure ESR of 18 samples in
– Technical factors: High temperature, tilting of 10 minutes, which is a very short period compared to the
ESR tube, Vibration of tube during test Westergren method, which takes 60 minutes for ESR
● Factors decreasing ESR measurement.
– Microcytosis
– Low fibrinogen
– Polycythemia
– Marked leukocytosis

SOURCES OF ERROR
1. If the concentration of anticoagulant is increased, the ESR
will be falsely low as a result of sphering of the RBCs, which
inhibits rouleaux formation.
2. The anticoagulants sodium or potassium oxalate and heparin
cause the red blood cells to shrink and falsely elevate the
ESR.
3. A significant change in the temperature of the room alters
the ESR.
4. Even a slight tilt of the pipette causes the ESR to increase.
5. Blood specimens must be analyzed within 4 hours of
collection if kept at room temperature (18 to 25° C).
6. Bubbles in the column of blood invalidate the test results.
7. The blood must be filled properly to the zero mark at the
beginning of the test.
8. A clotted specimen cannot be used.
9. The tubes must not be subjected to vibrations on the lab
bench which can falsely increase the ESR.
10. Hematologic disorders that prevent the formation of rouleaux
(e.g., the presence of sickle cells and spherocytes) decrease
the ESR.
11. The ESR of patients with severe anemia is of little diagnostic
value, because it will be falsely elevated.

AUTOMATED ESR METHOD

VES- MATIC 20 instrument.

● Requirements
● 7.5-cm heparin-free capillary tube, with an internal
and an external diameter of 1.1mm and 1.5mm.
● 3.2% trisodium citrate
● Plasticine tray having modeling clay or sealing
wax plates.
● Ruler

ESR STAT PLUS