Hematology (Midterm)

VITAMIN B12 AND FOLIC ACID

DEFICIENCY
Megaloblastic anemia
Proposed early in1960s as the
methylfolate trap
Characterized by defective
synthesis in DNA
Result from lack of folic acid or Vit.
B12
Macrocytic anemia(large RBCs in
the circulation)
Associated with ineffective
erythropoiesis
MCV> 100 fl but normal MCHC
Macrocytic or normochromic
Presence of megaloblast in BM and
macrocytic
o Due to the defective DNA
synthesis
Vit B12(cobalamin and
folate deficiency
Vitamin B12
o Essential nutrient consisting
of tetrapyrrole(corrin) ring
containing cobalt.
o Transfer of methyl group
from 5-methyl THF to
homocysteine
o Sources
Diary product( fish,
eggs, meat)
No Vit. B12 is present
in vegetables
Folic acid or folate
o Used for any form of vitamin
folic acid
o Synthetic form in
supplement and fortified
food
o Plays as an important role in
metabolism of amino acids
and nucleotides
o Circulate in the blood as
methyl folate
o Source: green vegetables,
yeast and cereals
Vit B12 Absorption
o Vit B12 transport is
mediated by three different
binding protein that are
capble of vinding the vitamin
at its required physiological
conc
Intrinsic factor(IF)
forms a protective
complex with B12that
is transported in the
GI tract
o Transcobalamin II (TCII)a beta- globulin produced by
the liver and the dominant
carrier of B12 after
absorption. Also the main

agent for rapid transport of

Vit. B12.
o R (rapid) Proteins/ Rbinders
o Transcobalamin I (TCII) an alpha- globulin produce
by granulocytes. A circulatin
reserve store (methyl
cobalamin) of B12.
o Transcobalamin III (C III)
also an alpha-globulin and
may act as a defence by
depriving pathogens of B12.
Two Mechanisms
o Active (75%)- requires the
presence of intrinsic factor (a
glycoprotein produced by
the mucosa).
o Passive occurs by diffusion
and works when
pharmacological doses of
vitamin B12 are ingested.
Storage sites
o Total amount of vitamin in
the body is 2-5mg
o Major site: liver
o Excreted through the bile
and shedding of intestinal
epithelial cells
o Most excreted Vit. B12 is

again absorbed by the

intestine. (enterohepatic
circulation)
Signs and Symptoms
o fatigue
o muscle weakness
o loss of appetite/weight loss
o diarrhea
o nausea
o fast heartbeat
o smooth or tender tongue
o tingling in hands and feet
o numbness in extremities
Causes/ Individuals at Risk of
Megaloblastic Anemia
Age
o -Older people >60
Family History
Dietary Habits
Strict vegetarian who doesnt eat
any animal or dairy products and

Hematology (Midterm)
doesnt take a vitamin B12
supplement
Alcoholics
Laboratory Diagnosis
Screening Tests
o Complete Blood Count
Hb, Hct,RBCs, WBCs,
Platelets
MCV, MCH ( normal
MCHC )
o Morphology

Hypersegmented Neutrophils, oval

macrocytes, anisocytes, poikilocytes,
RBC inclusions (Howell Jolly, Cabot
Rings, Basophilic Stippling)
Absolute reticulocyte
count
decreased
Gastric Assay
For achlorhydia
(inability to produce
HCl) thus dietary
B12 cant be
sparated from the
protein in food.
Specific Test
Bone Marrow Examination
Asynchrony of Nucleus and
cytoplasm
Hypercellular, erythroppoietic
Activity, Giant metamyelocyte
Chemistry
Tets for serum B12 or serum folate
or RBC folate
Competitive Protein- Binding
Radioassay for B12 and Folate
Patients B12 and folate in serum
compete with Co-labeled
cobalamin and I-labeled folic acid
B12 binder: Hog IF
The Schilling Test
Measure viable IF and absorption of
B12
Urinary excretion of co-labeled folic
acid
To test the absorption of
radiolabeled B12 in the absence
and presence of orally
administered IF
IF Deficiency if 2nd test is normal,
B12 malabsorption if bothe tests
are abnormal.
Normally 7% is excreted of the
administered dose of Co-B12. 3%
for Pernicious Anemia
Specific Test
Deoxyuridine Suppression Test
o Very sensitive
o Defect in thymidine
synthesis either b12 or folate
deficiency
Urinary Excretion of
Methylmalonic Acid
o Specific test: because only
B12 together with a specific
2

enzyme converts
methylmalonic CoA to
succinyl CoA
X-ray or Endoscopic Studies
o This for suspected Pernicious
Anemia (PA)
Bone Marrow Changes
o Markedly hypercellular
o Myeloid: erythroid ratio
decreaesed or reversed
o Erytropoiesis:
MEGALOBLASTIC
PERNICIOUS ANEMIA
autoimmune disorder
gastric atrophy of parietal cells
Reduced secretion of IF (intrinsic
factor)
vitamin B12 deficiency
Etiology
Impaired RBC production caused
by:
Cobalamin deficiency may result
from the following:
o Inadequate dietary intake
o Atrophy or loss of gastric
mucosa
o Functionally abnormal IF
o Bacterial overgrowth in
intestine
Morphology and classification
Macrocytic Anemia
Improper blood cell
formation
Impaired DNA
synthesis
Vit. B12 deficiency
Lack of intrinsic factor
Inadequate diet
Loss of gastric
mucosa
Ileal dysfunction
Pathogenesis
Genetic factor
o Common blood group A
o Ancestry( Northern
European, Asean Ancestry)
o Eye color (blue), white skin
complexion
Autoimmune factor
o Pre-existing autoimmune
disease
o Ab against parietal cells & IF
Three types of Antibodies
Type 1- blocks vitamin B12 and IF
binding
Type 2- prevents binding of IF- B12
complex with ileal receptors
Type 3- against specific structures
in the parietal cell.
Clinical Signs & Symptoms

Hematology (Midterm)
Macrocytic anemia
Glossitis
Paleness of the skin
Fatigue
Shortness of breath
Peripheral neuropathy
degeneration of spinal cord
Paresthesia
o Tingling sensation due to
nerve dysfunction
Screening & Confirmatory Test
Ab against parietal cells & IF
Endoscopy
Serum B12 level- <160 ng/L
CBC Count
MCV- >100 fL
Bone marrow examination
Schilling Test
How is the test is performed?
o Includes four different stages
Stage I: two doses of vit. B12
First dose: radioactive
form of Vit.B12 (mouth)
Second dose: larger dose
by shot 1 hr later
Urine collection over the
next 24 hrs
Abnormal:
inability of PC to secrete IF
Stage I is done 3- 7 days
later
Stage II
First dose: Radioactive
VitB12 w/ IF
o Abnormal: Proceed to Stage
III
Stage III
Done after taking
Antibiotics for 2 weeks
o May cause due to VitB12
consumption by bacteria
Stage IV
Pancreatic enzyme for 3 days
Followed by radioactive dose
of VitB12
Normal Results
o A normal result would
require urinating eight to 40
percent of the radiolabelled
vitamin B12 within 24 hours.
Abnormal Results
o Abnormal stage one and
normal stage two indicates
the stomach is unable to
make intrinsic factor.
BM-Megaloblasts present
PS-Macrocytes are observed
o 6-lobed polymorphonuclear
leukocyte is present
NON-MEGALOBLASTIC MACROCYTIC
ANEMIA

Macrocytosis- due to the excess

RBC membrane occurs in patient
with chronic diseases.
o MCV of about 100-105 fL
can occur with chronic alcohol use
These are disorders in which the
macrocytosis is not primarily due to
vitamin B12 or folic acid deficiency
Here the macrocytes are
ROUND
Related to changes owing to
disruption of the cholesterol-tophospholipid ratio
Diagnosis
- CBC, RBC indices,
reticulocyte count, and
peripheral smear
- Hematocrit and
Hemoglobin
- Sometimes bone marrow
examination
- Absence of
hypersegmented
neutrophils and
macroovalocytes
PS- stomatocytes, reticulocytes
BM- Megaloblast
Iron deficiency
Etiology and Morphology
- develops when the intake of
iron is not enough to meet
the standard level
- impaired absorption
- there is a chronic loss of
hemoglobin in the body
- gastrointestinal tract
abnormalities

IRON DEFICIENCY ANEMIA

is characterized by variable degrees
of microcytosis (MCV <80 fl) and
hypochromia (MCHC <32 g/dl) which
results in widening of the central palor
accounting for >1/3 of the total RBC
diameter.
The variation in cell size results in
higher RDW reflecting the fact that
iron deficiency is acquired disorder
with different levels of hemoglobin
production in red cells.
Pathogenesis
Hemoglobin synthesis is impaired
as a consequence of reduced iron
supply.
There a defect in cellular
proliferation.
Survival of erythroid
precursors and
erythrocytes is
reduced

Hematology (Midterm)

Signs and Symptoms

Fatigue and other nonspecific
symptoms
Epithelial tissue
Immunity and infection
Pica
Genitourinary system
Skeletal system
Screening and confirmatory test used to
determine the type of anemia
Screening
Routine CBC
o If CBC findings show a
hypochromic, microcytic
anemia with an elevated
RDW but no consistent
shape changes to the RBCs
patient is suspected Iron
deficiency
o elevated RDW [RBC
distribution width]
o RBC count & hematocrit
decreases
As the hemoglobin level continues
to fall, microcytosis and
hypochromia become more
prominent, with progressively
declining values for mean cell
volume (MCV), mean cell
hemoglobin (MCH), and mean cell
hemoglobin concentration (MCHC).
Low absolute reticulocyte count-diminished rate of effective
erythropoiesis because this is a non
regenerative anemia
Confirmatory tests
o serum iron total iron-binding
capacity (TIBC)
transferrin saturation
serum ferritin

Serum iron is a measure of the

amount of iron bound to transferrin
(transport protein) in the serum.
Principle: Serum ferric iron is first
removed from attachment to
transferrin by the addition of a
chemical such as 0.05N
hydrochloric acid.

Measurement: Spectrophotometry
at 562 nm
Reference Range: 50-150 ug/dL
Male-125 um/dL
Female- 100 um/dL
TIBC
is an indirect measure of
transferrin and the available
binding sites for iron in the
plasma.
Principle: The concentration of
transferring is measured indirectly
by measuring the ability of serum
transferring to bind rion.
Measurement: Spectrophotometry
at 562 nm
Reference Range: 250-450 ug/dL
(adult)
Transferrin saturation may only
be obtained if serum iron and TIBC
values are available and computed.
Principle: The percent of transferrin
saturated with iron can be
calculated from the total iron and
the TIBC:
Transferrin saturation=

Serumiron
TIBC

X 100

Reference Range: 20-55%

saturation

Serum ferritin is an easily

accessible surrogate for stainable
bone marrow iron. The iron studies
are used collectively to assess the
iron status of an individual. Shows
that, as expected, serum ferritin
and serum iron values are
decreased in iron deficiency
anemia, a state called sideropenia.
Measurement: Radioimmunoassays
Reference range:
Men: 15-200 ug/L
Women: 12-150 ug/L
ADDITIONAL TEST
Free erythrocyte protoporphyrin is
an early indicator of an iron
metabolism disorder.
Measurement: Hematofluorometer
Reference Range: <50 ug/dL
Therapeutic trial of iron provides a
less invasive and less expensive
diagnostic assessment
Zinc protoporphyrin is assayed
fluorometrically
Peripheral blood film from a patient
with hypochromic, microcytic
anemia.(X1000)
Iron deficiency anaemia: peripheral
blood film showing hypochromic
microcytes, elliptocytes, pencil
cells and fragmented cells.

Hematology (Midterm)
(x1000)
Bone marrows smear from a
patient with iron deficiency
anemia. (x 1000)
Perls Prussian blue stain showing
hemosiderin in a fragment of bone
marrow. (x1000)

Vitamin B12 deficiency

Aplastic Anemia
Myelodysplastic syndrome
Erythroluekemia
Chronic Liver disease
folate deficiency and some drugs
B. Etiology

ANEMIA OF CHRONIC DISEASE

Anemia is commonly associated

with systemic diseases, including
chronic inflammatory conditions
such as rheumatoid arthritis,
chronic infections such as
tuberculosis or human
immunodeficiency virus infection,
and malignancies.
o

Cartwright was the first to

suggest although the
underlying diseases seem
quite disparate, the
associated anemia may be
from a single cause,
proposing the concept of
anemia of chronic disease.
The anemia represents the
most common anemia
among hospitalized patients.

Anemia of chronic
inflammation is the preferred
term since not all chronic
diseases are associated with
this form of anemia.

Cytometric classification

Normochromic, normocytic
anemia (normal MCHC, normal
MCV).
o These include:
o
o
o
o
o

o
o

Impaired Ferrokinetics

Diminished
erythropoiesis and a
blunted response to
erythropoietin

Impaired Ferrokinetics
First Acute Phase Reactant
o Role of Hepcidin
Levels increase during
inflammation
Hormone produced by
hepatocytes to
regulate body iron
levels
Interacts with
ferroportin
When body iron levels
decrease, hepcidin
production by
enterocytes decreases
Nonspecific defense
against invading
bacteria.
Second Phase Acute Reactant
o

Factors that Contribute to

the Anemia of Chronic
Inflammation

III. Mechanism or pathogenesis of the

anemia

iron deficiency anemia,

thalassemia
anemia of chronic disease
(rare cases)
Sideroblastic Anemia

Normochromic, macrocytic
anemia (normal MCHC, high
MCV).
o These include:

Three Pathogenic
Mechanism to explain the
development of anemia
during illness:

a.1 Impaired mobilization of iron

Low serum iron level
Decreased iron in marrow erythroid
precursor
Normal or increased iron in the
marrow store
a.2. Impaired marrow response to
anemia
Deficiency in erythropoietin level
Response of erythroid marrow to
erythropoietin may be reduced
a.3. Shortening of red cell survival
Mild reduction in RBCs life span as
a result of extra corpuscular factors
including overactive histiocyte
destruction of red cells.

Hypochromic, microcytic
anemia (low MCHC, low MCV).
o These include:
o

anemias of chronic disease

Infection (parvovirus B19)
anemia of renal disease
aplastic anemia
Myelophthisic anemia

Lactoferrin

Hematology (Midterm)
Iron binding protein in
the granules of
neutrophils.
Greater avidity in iron
than transferrin
Are important
intracellularly
Released in plasma
during inflammation

causing
a
normocyticnormochromic anemia with high
RBC distribution width or a
microcytic-hypochromic
anemia,
particularly with increased serum
iron and ferritin and transferrin
saturation.
a form of anemia in which the bone
marrow
produces
ringed
sideroblasts rather than healthy
red blood cells (erythrocytes).

In sideroblastic anemia, the

body has iron available but cannot
incorporate it into hemoglobin,
which red blood cells need to
transport oxygen efficiently.
Classification
Congenital sideroblastic anemia
Hereditary
sideroblastic
anemias
are
usually
small
(microcytic) and of poor color
(hypochromic) and thus must be
distinguished from iron deficiency
and thalassemia.

Third Acute Phase Reactant

o

Ferritin
Increased levels in the
plasma bind to some
iron
Unavailable for
incorporation into
hemoglobin
Effects: abundant in
bone marrow
macrophages

release to
developing
erythrocyte is slowed

Diminished erythropoiesis and a

blunted response to erythropoietin

IV. Definitive Signs and Symptoms

o
o

X-linked sideroblastic
anemia:
most common congenital cause of
sideroblastic anemia and involves a
defect in ALAS2, which is involved
in the first step of heme synthesis.
Although X-linked, approximately
one third of patients are women
due to skewed X-inactivation.
Autosomal recessive sideroblastic
anemia
involves
mutations
in
the
SLC25A38 gene. \
it is involved in mitochondrial
transport of glycine.
o Glycine
is a substrate for
ALAS2 and necessary
-

Anemia of chronic disease is

often mild. You may not
notice any symptoms.
When symptoms occur, they
may include:

Feeling weak or tired

Headache
Paleness
Shortness of breath

Anemia of Chronic
Inflammation

V. Screening and Confirmatory Tests to

determine the type of anemia

Peripheral blood picture

o Mild anemia
o Hemoglobin concentration9 to 11g/dL
o Without reticulocytosis
Cells are normocytic and
normochromic
o Microcytosis and
hypochromia develop in
about one third of patients
Leukocytosis and Thrombocytosis

Sideroblastic and Refractory Anemia

Sideroblastic anemias
are iron-utilization anemias that
are
usually
part
of
a
myelodysplastic syndrome,
6

Serum ferritin

Increased/normal

Serum iron

Decrease

TIBC

Decrease

Transferrin saturtion

Decrease/normal

FEP/ZPP

Increase

BM iron (Prussian
blue reaction)

Increase/normal

Sideroblasts in BM

None/very few

Other special tests

Specific tests for

inflammatory disorders
or cancer
for heme synthesis.
The
autosomal

Hematology (Midterm)
recessive
form
is
typically severe in
presentation.
Acquired clonal sideroblastic anemia
Clonal sideroblastic anemias fall
under the broader category of
myelodysplastic syndromes (MDS).
Three forms exist and include
refractory anemia with ringed
sideroblasts (RARS),
refractory anemia with ringed
sideroblasts and thrombocytosis
(RARS-T),

refractory
cytopenia
with
multilineage dysplasia and ringed
sideroblasts
(RCMD-RS).
These
anemias
are
associated
with
increased
risk
for
leukemic
evolution.

hereditary sideroblastic anemias

result from mutations in the gene
encoding ALA-S.
Definitive Signs and Syptoms
- Skin paleness, Fatigue,
Dizziness,
Enlarged
spleen and liver
Screening and Confirmatory Tests
Routine laboratory testing may be
used to discover incidental disease.
Genetic
testing
provides
confirmation of the diagnosis for
most patients with hereditary
hemochromatosis.
Liver biopsy with assessment of
iron staining and degree of scarring
in liver specimens is essential to
determining the degree of organ
damage.

Acquired
reversible
sideroblastic
anemia
Causes include excessive alcohol
use (the most common cause of
sideroblastic anemia), pyridoxine
deficiency, lead poisoning, and
copper deficiency.
Excess zinc can indirectly cause
sideroblastic anemia by decreasing
absorption and increasing excretion
of copper.

Antimicrobials that may lead to

sideroblastic
anemia
include
isoniazid,
chloramphenicol,
cycloserine, and linezolid.
Pathogenesis
The
process
begins
in
the
mitochondrion
with
the
condensation
of
glycine
and
succinyl-CoA to form delta-amino
levulinic acid (ALA). Pyridoxal
phosphate is a cofactor in the
reaction. ALA then moves to the
cytoplasm where several additional
enzymatic transformations produce
coproporphyrinogen
III.
This
molecule enters the mitochondrion
where additional modifications,
including the insertion of iron into
the protoporphyrin IX ring by
ferrochelatase,
produce
heme.
Defects in the cytoplasmic steps of
heme biosynthesis cause various
forms of porphyria. Functional
abnormalities
of
the
enzyme
porphobilinogen deaminase, for
instance,
produce
acute
intermittent porphyria.
In contrast, defects in the steps of
heme biosynthesis that occur
within the mitochondrion produce
sideroblastic anemias. For instance,
perturbations of the enzymatic
activity of delta-amino levulinic
acid synthase (ALAS) produce
sideroblastic anemia. The X-linked

Blood Picture
o Peripheral blood smear
showing many hypochromic
and microcytic cells.
o Bone marrow smear showing
erythroid hyper plasia,
erythroblasts with defective
hemoglobinization and
erythroblasts containing
multiple Pappenheimer
bodies and most erythroid
precursors are ring
sideroblasts.

Hematology (Midterm)
PORPHYRIAS
-diseases characterized by impaired
production of heme.
-may be:
Acquired Porphyria Lead Poisoning
Inherited Porphyria deficiency in
enzymes in the production of heme
pathway
-can cause Sideroblastic Anemia
(deficiency in the enzyme Aminolevulinic
Acid (ALA) Synthethase, ALA dehydratase,
Ferrochelatase.

HEMOCHROMATOSIS and
HEMOSIDEROSIS
Hemosiderosis-is focal deposition of iron
that does not cause tissue damage.
*Transfusion-related
hemochromatosis-when there is a
repeated transfusion for anemic
patients, and the iron from the RBC
will be added to the normal 1 mg/day
that will be stored in the body.
Hemochromatosis (iron overload)- is a
typically systemic process in which iron
deposition can cause tissue damage.

-Clinical Sign and Symptoms:

Port Wine Red Urine (porphyrin in
urine)
Skin is very sensitive to light,
especially direct sunlight (fluorescent
products o
Eyes may also be sensitive to bright
sunlight
Skin may take longer to heal after
injury or blistering
Anemia
Splenomegaly

*Erythroblast will appear bright red in

fluorescence microscope
*Erlichs Test for the detection of Acute
Intermittent Porphyria which uses Ehrlich's
aldehyde reagent consists of p-dimethyl
amino benzaldehyde in acid solution
*Ringed Sideroblast, Sideroblast and
Siderocytes is present in the blood smear.

Pathogenesis
1. Formation of Hemosiderin
-inactive metabolite and a result of
degeneration of ferritin inside the cell
2. Accumulation of Free Iron
(Ferrous)
-Ferrous in the presence of oxygen will
react to form Superoxides and Free
radicals
3. Peroxidation of All kinds of
Membranes
-Superoxides and Free radicals will
breakdown membranes (Cell
membrane, lysosomal membrane,
Mitochondrial membrane, etc.) leading
to the death of the cells.
Clinical Signs and Symptoms
Golden Color Skin-hemosiderin
deposition
Cirrhosis-induced Jaundice in the
Liver-sometimes it could lead to
cancer
Bronze diabetes-Hemochromatosis
in diabetic patients (pancreas is
affected)
Congestive Heart Failure
Tests
Transferrin Saturation-Screening
Test
Genetic Testing-Confirmatory Test
*Serum Ferritin is elevated in
hemochromatosis
Treatment

Hematology (Midterm)
Withdrawal of Blood-for hereditary
hemochromatosis patients
Iron-chelating drugs
(Desferrioxamine)
-bind with excess iron then eliminated
in order for the iron to not accumulate
in the plasma

SICKLE CELL ANEMIA

Hemoglobin S (2 26GluVal )
-congenital chronic hemolysis
-substitution of Glutamate to Valine in the 6th
Amino acid of the Beta Globin Chain
-Glutamate has a charge of (-1) and Valine
has a charge of (+0), a change of (+1)
charge, so thats why Hemoglobin S migrate
to other area in electrophoresis
-Glutamate is polar (hydrophilic), Valine is
non-polar (Hydrophobic)
-has a Hb S polymer that is long and slender
-low oxygen tension leads to polymerization
of Hemoglobin S
Sickle Cells
Reversible Sickle Cells-Hb Scontaining erythrocytes that change in
shape in response to oxygen tension.
-normal biconcave when oxygenated,
and changes in shape when
deoxygenated
-causes vassoocclusive complication
(can pass through the
microvasculature because it is normal
biconcave when oxygenated, and it
will change shape in tissues because it
will be deoxygenated)
Irreversible Sickle Cells-do not
change in shape whether it will be
oxygenated or not.
-Elongated sickle cells with a point at
each end
-removed by the spleen and there is a
less chance that it will enter the
microcirculation and cause
vasoocclusions.
Intracellular Hydration
-Polymerized deoxyhemoglobin activates
Psickle membrane channel which decreases
the oxygen to less than 50 mm Hg and
allowing the influx of Ca2+ which will activate
second membrane channel called Gardos
Channel
-Gardos channel stimulates the efflux of K+
and Cl-, water efflux will then follow to
maintain equilibrium

-Water efflux causes dehydration of the cell,

thereby the HbS concentration is higher, so
as a result polymerization of HbS is intensify
Pathogenesis
1. Hb S in RBCS is less soluble forming
tactoids/liquid crystals that causes
sickling
2. Blood becomes more viscous when
sickle cells and polymers are formed
3. Increase Blood viscosity cause
decrease blood flow.
4. Increase blood flow prolongs the
exposure of Hb S-containing
erythrocytes to a hypoxic
environment.
5. Hypoxic environment lowers pH which
decreases oxygen affinity, which
further promotes sickling
6. Occlusion of capillaries and arteries by
sickled RBCs and infarction of
surrounding tissues
Clinical Signs and Symptoms
Vasoocclusion-main problem
Splenomegaly due to splenic
sequestration
Episodes of Pain
Acute chest syndrome (fever, chest
pain and presence of pulmonary
infiltrates
Pulmonary hypertension-fatal
Tests
Peripheral Blood Smear
*Wright stain is used and the presence
of a long, curved cell with a point at
each end is present.
*Target cell is also present
Old Screening Test
*addition of sodium metabisulfite (a
reducing agent) on a slide with a drop
of blood, the mixture is sealed under a
coverslip. Hemoglobin is reduced into
its deoxygenated form, then it
promote polymerization of HbS (if HbS
is present), the morphology of the cell
will change, and it is examine
microscopically.
Hemoglobin Solubility Test
-reagent used: Saponin and Sodium
dithionite
*Saponin-promotes lysed of RBC,
releasing Hb
*Dithionite-react with Hb converting it
to its deoxygenated form
-Deoxygenated HbS is insoluble
and precipitates in the solution
which causes turbidity. Normal Hb
is clear
Electrophoresis
*Cellulose Acetate-co-migrate with
Hb D and G

Hematology (Midterm)
*Citrate Agar-migrate alone after
Hb C
Treatment
-Supportive Treatment
Neonatal screening
Chilhood prophylactic penicillin
theraphy
Bone marrow transplantation
Treatment with hydroxyurea
*Sickle cell trait is resistance against
infection with Plasmodium falciparum.

10

Hemoglobin C-Harlem (C-Georgetown)

-double substitution , 6th AA, Glutamate to
Valine, 73rd AA, Asparagine to Aspartate
-Positive in Hemoglobin Solubility Test
Hemoglobin C (22 6GluLys)
-Has hemoglobin C Crystal, also known as
gold bar crystals (hexagonal crystals)
-produce mild hemolytic anemia
Hemoglobin E (22 26GluLys)
Hemoglobin D (22 121GluGln)
Hemoglobin G (2 68AsnLys 2)
Hemoglobin O-Arab (22 121GluLys)

Hematology (Midterm)

11

Hematology (Midterm)
UNSTABLE HEMOGLOBIN VARIANTS
-results from genetic mutations to globin
genes creating hemoglobin products that
precipitate in vivo, producing Heinz bodies
causing a hemolytic anemia.
-majority of these variants are chain
variants
-before it was called Congenital non
spherocytic hemolytic anemia or
Congenital Heinz body anemia
-properly called Unstable Hemoglobin
disease
-all patients are heterozygous because
homozygous is not suitable for life
-most prevalent unstable hemoglobin is Hb
Kln
Causes of Hemoglobin Variants
1. Substitution of a charged for an
uncharged amino acid in the interior of
the molecule
2. Substitution of a polar for a non polar
amino acid in the hydrophobic heme
pocket
3. Substitution of an amino acid in the
and chains at the intersubunit
contact points
4. Replacement of an amino acid with
proline in the helix section of a chain
5. Deletion or elongation of the primary
structure
Clinical Signs and Symptoms
Hemolytic Anemia
Jaundice
Splenomegaly
Dark Urine (contains dipyrrole)
Tests
Peripheral Blood Smear
-prominent basophilic stippling
-Supravital stains woul dhow Heinz
bodies (larger and numerous)
Electrophoresis
-not detected because it migrates
together with normal AA pattern
Isopropanol Precipitation Test
-Isopropanol @ 37 degrees C weakens
the bonding forces of the hemoglobin
molecule. If unstable hemoglobins are
present, rapid precipitation occurs in 5
minutes, heavy flocculation occurs
after 20 minutes.
*Normal hemoglobin does not
precipitate until approximately 40
minutes.
Heat denaturation Test
-incubation @ 50 degrees C for 1 hour.
*Unstable hemoglobin show a
flocculent precipitation
*little or no precipitation for normal
hemoglobin

12

High Performance Liquid

Chromatography
DNA-based globin gene analysis

Treatment
*In severe cases, Spleen must be remove to
reduce sequestration and rate of removal of
RBCs

HEMOGLOBINS WITH INCREASE AND

DECREASED OXYGEN AFFINITY
Recall:
Deoxygenated Hemoglobin
-Tensed state
-low affinity for heme ligand: oxygen
-high affinity for Bohr protons and 2, 3BPG
Oxygenated Hemoglobin
-Relaxed state
-high affinity for heme ligand: oxygen
-low affinity for Bohr protons and 2, 3BPG
Hemoglobins with Inceased Oxygen
Affinity
-Hb Abruzzo and Hb Crete
-failed to release oxygen resulting to hypoxia
-hypoxia triggers kidney to release
erythropoietin, which leads to compensatory
erythropoiesis
-do not precipitate in vivo to produce
hemolysis
-no abnormal RBC morphology
-detected by Electrophoresis using Citrate
agar (pH 6.0) or by gel electrophoresis.
-Oxygen Affinity measurement for definitive
diagnosis
-patients are usually normal and dont
require treatment
Hemoglobin with Decreased Oxygen
Affinity
-Hb Kansas
-quickly release oxygen to the tissues, which
results in normal to decreased hemoglobin
concentration and slight anemia
-detected by Starch gel electrophoresis,
HPLC, or DNA-based globin gene
analysis

THALASSEMIA
-group of heterogeneous disorders in which
one or more globin chains are reduced or
absent

Hematology (Midterm)
o

o
o

Silent Carrier State (silent/)

-caused by one mutated gene that
produces a small decrease in chains.
-normal blood picture
-Thalassemia Minor
-mild, asymptomatic, microcytic,
hypochromic anemia
-elevated HbA2 level
-Thalassemia Major
-severe anemia leading to transfusion
dependence
-Thalassemia Intermedia
-manifest abnormalities with a
severity between those of Thalassemia Major and -Thalassemia
Minor

*Hb Lepore thalassemia

-hereditary persistence of fetal
hemoglobin

-results to hypochromic, microcytic anemia

due to decreased production of hemoglobin
ALPHA THALASSEMIA
-caused by a deletion of one or both of the
genes on chromosome 16, resulting in
reduced or absent production of chains
-tetramers of chains may precipitate as Hb
Bart in the fetus and newborn
-tetramers of chains may precipitate as Hb
H in the adult
-divided into:
o Silent carrier -thalassemia
-result of the deletion, or rarely a non
deletional mutation, of one of four
genes (-/) or (T/)
-associated with a normal RBC profile
and is asymptomatic
o -Thalassemia minor
-result of the deletion of two genes
(-/-) or (--/)
-mild, asymptomatic, microcytic,
hypochromic anemia, HbA2 is not
elevated
o Hb H disease
-result of the deletion of three of the
four genes (--/-)
-HbH inclusions (excess chains)
precipitate in older circulating RBCs,
causing a hemolytic anemia
-RBCs are microcytic and
hypochromic, similar to -thalassemia
intermedia
o Hb Bart hydrops fetalis
-result of the deletion of four of the
genes (--/--)
-severe anemia in utero
-incompatible with life
BETA THALASSEMIA
-clinically manifested as:

13

TESTS (for both and Thalassemia)

o Complete Blood Count
-hemoglobin and hematocrit are
decreased
-low MCV, MCHC
-RBC Distribution Width (RDW) is
elevated
o Reticulocyte Count-elevated
o Bone Marrow Examination
o Osmotic Fragility Test-decreased
o Supravital Staining
*HbH inclusion-greenish blue bodies
o Electrophoresis

APLASTIC ANEMIA
-is a rare but potentially fatal bone marrow
failure syndrome.

Hematology (Midterm)

Acquired Aplastic Anemia

-classified as idiopathic (70%) when the
cause is unknown and as secondary (10%)
when the cause is identified
-pancytopenia is common (decrease blood
cells)
Inherited Aplastic Anemia
-show manifestations of the disorder at an
early age and may have physical
malformations.
o

o
o

14

Fanconi Anemia
- Chromosome instability disorder
characterized by aplastic anemia,
physical abnormalities, and cancer
susceptibility.
Dyskeratosis Congenita-bone
marrow failure
Shwachman-Diamond Syndrome
-pancreatic insufficiency, cytopenia,
skeletal abnormalities, and a
predisposition for hematologic
malignancies.

Hematology (Midterm)
PURE RED CELL APLASIA
-is a rare disorder of erythropoiesis
characterized by a selective and severe
decrease In erythrocyte precursors in an
otherwise normal bone marrow.
-characterized by severe anemia
(normocytic), reticulocytopenia, and normal
RBC and platelet count.
Acquired Pure Red Cell Aplasia
-can be categorized into:
Primary PRCA-may be idiopathic or
autoimmune related.
Secondary PRCA-may occur in
association with an underlying
thymoma, hematologic malignancy,
solid tumor, infection, chronic
hemolytic anemia, collagen vascular
disease, or exposure to drugs or
chemicals.
-in young children it is also known as
Transient eryhtoblastopenia of
childhood
-Transfucions are initial therapy
Congenital Pure Red Cell Aplasia:
Diamond-Blackfan Anemia
-A congenital erythroid hypoplastic disorder
of early infancy
-mutations in genes that code for structural
ribosome proteins
-Mutations in these ribosomal proteins
disrupt ribosome biogenesis in DBA
-macrocytic anemia with reticulocytopenia
-normal WBC and platelets
-treatment is RBC transfusion and
corticosteroids

CONGENITAL DYSERYTHROPOETIC
ANEMIA (CDA)
-heterogeneous group of rare disorders
characterized by refractory anemia,
reticyclopenia, hypercellular bone marrow
with markedly ineffective erythropoiesis, and
distinctive dysplastic changes in bone
marrow erythroblasts.
-classified into:
CDA I

15

CDA II
CDA III

MYELOPHTHISIC ANEMIA
-infiltration of abnormal cells into the bone
marrow and subsequent destruction and
replacement of the normal hematopoietic
cells.
-Cytokines, growth factors, and other
substances are releases that suppress
hematopoiesis or destroy stem, progenitor,
or stromal cells, which results in peripheral
cytopenias.
-if the infiltration and proliferation of the
abnormal cells disrupts the normal bone
marrow architecture, premature release of
immature cells from the bone marrow occurs.
-mild to moderate with normocytic
erythrocytes and reticulocytopenia.
-presence of teardrop erythrocytes and
nucleated RBCs, but immature myeloid cells
(leukoerythroblastic peripheral blood
picture), megakaryocyte fragments, and
giant platelets also may be present

ANEMIA OF CHRONIC KIDNEY DISEASES

-anemia occurs in most patients with chronic
kidney disease
-anemia caused by inadequate production of
erythropoietin by the kidneys
-anemia can also be caused by Uremic toxins
accumulate in the blood because of the
kidney failure, and they inhibit erythropoiesis
and shorten the life span of the RBCs
-normocytic , normochromic Red blood cells