RBC DISORDER: ANEMIA

Stephen Brian G. Valencia

 Disorder Characterized by Decreased
Red Cell Concentration: ANEMIA
1. Anemia of Bone Marrow Failure and Systemic Disorders
2. Anemia of Abnormal Nuclear Development
3. Anemia of Abnormal Iron Metabolism
4. Anemia of Abnormal Globin Development:
Hemoglobinopathies
5. Anemia of Abnomal Globin Development:Thalassemia
6. Hereditary Anemias of Increased Destruction
7. Acquired Nonimmune Anemia of Increased Destruction
8. Acquired Immune Anemia of Increased Destruction
9. Anemia of Blood Loss
 ANEMIA
• REDUCED Hb concentration of less than 12g/dL in
men and 11g/dL in women
• If the Hemoglobin or Hct is below the lower limit of
the 95% reference interval for individual’s sex, age,
and geographical location

Clinical symptoms:
- Decreased oxygen-carrying capacity of the blood and
tissue hypoxia
What will be the response of the body?

Low in O 2
Low tissue
Decrease RCM carrying oxygenation
capacity

Growth and Kidney release

Maturation of
EPO hypoxia
RBC

Increase RPI;
Increase retics;
(Shift retics)
4
 Physiologic effects:
1. Decreased Hb oxygen-affinity state = shift to
the right
2. Decreased tissue perfusion(selective
redistribution of blood flow)
3. Increased cardiac output (cardiac stress)
4. Increased RBC production by the marrow:
- erythroid hyperplasia
-increased reticulocyte count
-nucleated RBC in the circulation
 Clinical Manifestations of Anemia

• Asymptomatic
• Palpitations & dyspnea during exercise
• Increased cardiac stress may cause
– Tachycardia
– Shortness of breath
– Headache
• Pallor
– Dermal vasoconstriction and blood redistribution
• Secondary to tissue hypoxia
– Leg cramps
– Dizziness
– Fatigue
– Insomnia
• Coma
• Death
 Relative vs Absolute Anemia
RELATIVE ANEMIA
• Fluid shift from extravascular
to intravascular
• Normal red cell mass
• Plasma volume increased
• Secondary to an unrelated
condition and can be transient
in nature
• Common cause is
hemodilution
– Pregnancy
– Volume overload
ABSOLUTE ANEMIA
• Cause by factors that leads
to anemia
• True decrease in red cell
mass
• Plasma volume is normal
• Impaired RBC production
• Blood loss
• Accelerated RBC destruction
 Relative Anemia
A. Dilutional anemia can occur by the third
trimester pregnancy
- compensatory increase in plasma volume

B. Macroglobulinemia and Multiple myeloma

- increase in plasma globulin and protein
concentration: hyperosmotic plasma
 Categories of Anemia
I. Hypoproliferative
– Eg. Aplastic anemia
II. Maturation disorder
– Eg. IDA, Vit. B12 Def
III. Hemolytic disorder
– Eg. Autoimmunity, HDN
IV. Blood loss
– Eg. Duodenal ulcers
 Classification of Anemia
1. Etiologic
2. Morphologic
3. Physiologic
4. Combine morphologic and physiologic
 Etiologic classification
1. Defective Hgb synthesis
2. Associated with Vit. B12 or folate
3. Impaired bone marrow or stem cell fxn
4. Decreased red cell survival and increased red cell
destruction
– Intrinsic cell defect
– Extrinsic red cell defect
5. Secondary to blood loss
6. DNA disorder
 Morphologic classification
1. Normocytic, normochromic
2. Microcytic, hypochromic
3. Microcytic, normochromic
4. Macrocytic, normochromic
 Physiologic classification
• Use of Reticulocyte Production Index (RPI)
– To determine ineffective and effective
erythropoiesis
• a.) Ineffective
– Hypoproliferative
– Maturation disorder
• b.) Effective
– Hemolytic anemia
– Blood loss
Combine Physiologic and Morphologic
Classification

1.) RPI
2.) Morphology
3.) RDW
 MORPHOLOGIC CLASSIFICATION OF
ANEMIA through RBC INDICES RANGE
RBC Morphology MCV MCH MCHC Anemias
(fL) (pg) (%)
Normocytic/ 80-100 27-31 32-36 •Acute blood loss
Normochromic •Hemolytic Anemias
•Aplastic Anemia (early)
•Myelopthisic anemia
•Stem cell related anemia
Macrocytic/ >100 >31 Normal •Megaloblastic
Normochromic •Anemia Anemia of liver
disease
•Chronic Aplastic anemia
•Acute hemolytic anemia
(w/ shift reticulocytosis)
RBC MCV MCH MCHC Anemias
Morphology (fL) (pg) (%)
Microcytic/ <80 27- 32-38 •Anemia of chronic
Normochromic 31 •inflammation
Microcytic/ <80 <27 <32 •Thalassemia
Hypochromic •Porphyria
•IDA
•Lead poisoning
•Sideroblastic
anemia
 I. Hypoproliferative Disorder
• Impaired bone marrow and stem cell function.

APLASTIC
ANEMIA

SYSTEMIC DISEASE,
MYELOPHTHISIC RENAL AND
ANEMIA ENDOCRINE

PURE RED
CELL APLASIA
A. APLASTIC ANEMIA
- Reduction in the number or function of
multipotential stem cells, resulting
pancytopenia.
- The disease is characterized by peripheral
pancytopenia and accompanied by a
hypocellular bone marrow.
- A severe, life threatening syndrome that may
occur in all age groups and both genders.
- Increased fat cell infiltration
HYPOCELLULAR BONE MARROW IN
APLASTIC ANEMIA
 APLASTIC ANEMIA
• Pathophysiology:
– The primary defect is a reduction in or depletion of
hematopoietic precursor stem cells with decreased
production of all cell lines. This is what leads to the
peripheral pancytopenia.

• This may be due to quantitative or qualitative

damage to the pluripotential stem cell.
• In rare instances it is the result of abnormal
hormonal stimulation of stem cell
proliferation
• or the result of a defective bone marrow
microenvironment
• or from cellular or humoral
immunosuppression of hematopoiesis.
Aplastic Anemia can be classified based on its
etiology:
1. Primary – congenital disorder

2. Secondary – acquired disorder

Primary Aplastic Anemia
Congenital disorders
Fanconi’s anemia
– the disorder usually becomes symptomatic ~ 5 years of
age and is associated with progressive bone marrow
hypoplasia.
- Congenital defects such as skin hyperpigmentation and
small stature are also seen in affected individuals.
- Autosomal recessive trait
With multiple chromosomal abnormalities
– Ring chromosome
– Translocation
– Breaks
– Dicentric form
- Can lead to Leukemia AML
Fanconi’s Anemia
Clinical manifestations:
Fatigue
Heart palpitations
Pallor
Infections
Petechiae
Mucosal bleeding
Microencephaly
Brown skin pigmentation
Short stature
Thumb malformation
“Cross eyed”
Malformation of kidney
Genital hypoplasia
Clinical features
Familial aplastic anemia
– a subset of Fanconi’s anemia in which the
congenital defects are absent.
 Secondary Aplastic Anemia
Acquired
Most cases of aplastic anemia are idiopathic and there is
no history of exposure to substances known to be
causative agents of the disease

 Exposure to ionizing radiation – hematopoietic cells

are especially susceptible to ionizing radiation. Whole
body radiation of 300-500 rads can completely wipe
out the bone marrow. With sublethal doses, the bone
marrow eventually recovers.

 Chemical agents – include chemical agents with a

benzene ring, chemotherapeutic agents, and certain
insecticides.
Idiosyncratic reactions to some commonly
used drugs such as chloramphenicol or
quinacrine.

Infections – viral and bacterial infections such

as infectious mononucleosis, infectious
hepatitis, cytomegalovirus infections, and
miliary tuberculosis occasionally lead to
aplastic anemia
Pregnancy (rare)
Other diseases – preleukemia and carcinoma
Aplastic Anemia
Lab findings:
1. Severe pancytopenia with relative lymphocytosis
(lymphocytes live a long time)
2. Normochromic, normocytic RBCs (may be slightly
macrocytic)

3. Mild to moderate anisocytosis and poikilocytosis

4. Decreased reticulocyte count

5. Hypocellular bone marrow with > 70% yellow marrow

Treatment – in untreated cases the prognosis is
poor
• Remove causative agent, if known
• Multiple transfusions
• Bone marrow transplant
 B. PURE RED CELL APLASIA
- Selective depletion of only the erythroid bone
marrow tissue
- Unipotential (CFU-E) stem cell defect related
to an immunologic dysfunction
- WBCs and platlets are unaffected.
- CLASSIFIED INTO:
1. Congenital: Diamond-Blackfan
2. Acquired: Primary & Secondary
 Diamond-Blackfan syndrome
- Congenital Pure Red Cell Aplasia
– occurs in young children and is progressive. It is probably
due to an intrinsic or regulatory defect in the committed
erythroid stem cell.
- RBC precursor cell unresponsive to EPO
- low reticulocyte count
- increase in 5%-25% Hb F
- presence of RBC fetal antigen
- Lymphocyte inhibits RBC maturation
Clinical presentation:
Anemia
Abnormal thumb
Retarded growth
Bone age retardation
Osteoporosis
Failure of 2nd sexual maturation
Acquired Pure Red Cell Aplasia
– Primary
• Idiopathic
• Immune mechanism ( antibodies against RBC AG and
EPO)
– Secondary
• Patients with thymoma – T-cell mediated responses
against bone marrow erythroblasts or erythropoietin
are sometimes produced.
• drugs, chemical, infection, HA
 Fanconi’s vs Diamond Blackfan
Fanconi’s Diamond-Blackfan
PBS Pancytopenia Low RBC
Bone Marrow Panhypoplasia Decreased RBC
Aspiration and Precursors
Biopsy
Cytogenetics Abnormal No known abnormality
chromosome
 Similarity of Fanconi’s and Diamond-
Blackfan
• Increased in EPO
• Normocytic, normochromic
• Low Hgb A1
• Increased Hgb F
 C. MYELOPHTHISIC ANEMIA
- Bone marrow infiltration and
hyperproliferation by nonerythroid cells
- bone marrow is replaced by abnormal cells
like tumor, cancer and fibrous tissue

a. Leukoerythroblastic reaction
b. Metastatic carcinoma
c. Multiple myeloma
d. Lymphoma
e. Leukemia
f. Lipidoses or storage disease
 Myelopthisic Anemia
• Laboratory:
-Normocytic,
normochromic
- Leukoerythroblastic
blood picture
 D. SYSTEMIC DISEASE
1. Anemia of Renal Disease
– normocytic normochromic;
– decreased reticulocytes count
–  EPO -  erythroid committed precursors
– effects of uremic toxins  decrease red cell
survival
*HUS – BURR CELL and RBC fragments
2. Anemia of Endocrine disorder
– may lead to decreased erythropoietin
production.
For example:
hypothyroidism leads to decreased demand
for oxygen from tissues; decreased androgens
in males; decreased pituitary function
ANEMIA OF ENDOCRINE DISEASE
a.) Anemia of HYPOTHYROIDISM
-thyroid regulates the cellular metabolic rate
and tissue oxygen requirement
- decrease in the production of EPO

b.) Deficiency in Testosterone Secretion

-in men results in a decrease in RBC
production and a decline of approximately
2g/dL of Hb.
 II. MATURATION DISORDER
1.) Abnormal nuclear development
a.) Vit B12 deficiency (Megaloblastic anemia)
b.) Folate deficiency (megaloblastic anemia)
c.) Congenital dyserythropoietic anemia
2.) Failure of Iron Metabolism
a.) IDA
b.)Sideroblastic anemia
ABNORMAL NUCLEAR DEVELOPMENT
- Megaloblastic type of anemia
- Defective DNA synthesis due to abnormal
nuclear maturation
- Nucleus mature smaller than the cytoplasm
(asynchronism)
- Oval RBC
- MCV is increased
 Vitamin B12 and Folate
Vit B12 Folate
– Belong to family cobalamin – Derived from pteroyl-
glutamic acid
– Important in DNA synthesis – Important in purine
– Eggs, meat, fish, milk and pyrimidine
synthesis
– Not found in veggies and fruit
– Green leafy veggies,
– RDA = 3 ug/day liver, kidney, cereals,
4 ug/day yeast, oranges
– RDA = 50ug or 400 ug
food folate
 Megaloblastic Anemia
Vitamin B12 deficiency
- can cause pernicious anemia
• Pernicious anemia (PA)
– Lack of IF, antibodies to intrinsic factor, or
antibodies to parietal cell.
• Gastritis ( congenital PA; juvenile PA )
• Mucosal damage
• Autoimmune ( thyroid disease)
• Endocrine disorder (thyroid, adrenal or pancreatic)
• Genetic disorder
Intrinsic factor- secreted by parietal cell and is needed to
bind vit B12 for absorption into the intestine.
Other Cause of Vitamin B12 deficiency
1.) Castle’s IF deficiency
2.) IF molecular defect
3.) Small bacterial overgrowth
4.) Fish tapeworm infection
5.) Ileal disease
6.) Ethanol and neomycin intoxication
7.) Achlorhydria
8.) Immerslund syndrome
– Autosomal recessive
– No receptors for IF-vit b12 complex; no problem in gastric
secretions
 Megaloblastic Anemia

• Folic Acid Deficiency

- Due to Dietary intake
- Alcoholic cirrhosis
- Pregnancy
- Infant malnutrition ( 50 ug/day )
• Folate antagonist
– Antimalarial drugs and methotrexate agent
• Inhibits dihydrofolate reductase
 Cause of both folate and Vit B12 def.
1.) Tropical sprue
– Atrophy of the entire small intestine
2.) Gluten-Sensitive enteropathy
– Affects the proximal part of the intestine
– patients react violently with gluten protein
 Clinical findings

• Glossitis ( beefy red)

• Paresthesias ( myelin degeneration)
• Difficulty in walking
• Anorexia
• Abdominal discomfort
• Lemon-yellow color
• Neurologic disturbance ( “megaloblastic
madness”)
Beefy red tongue
 Laboratory diagnosis
• Peripheral blood
– Macrocytes/ovalocytes
– MCV and MCH is increased
– MCHC is normal
– Decrease WBC, platelet count, retic count
– Hypersegmented neutrophil
– Presence of Howell-jolly, cabot ring, basophilic
stippling
– Bizarre poikilocytes ( hand mirror, dumbell etc.)
Morphologic Expression of Megaloblastosis

Peripheral smear
• Increased MCV with
macro-ovalocytes
associated with
anisocytosis and
poikilocytosis
• Hypersegmentation of
PMNs
• Thrombocytopenia
 Laboratory diagnosis
• Clinical chemistry
– Vit B12 level decrease
– LD 1 increased
– Iron overload
Tests:
• Schilling’s test – measures the ability to
secrete viable IF and absorption adm. Co-
labeled vit B12 in ileum
» >7% excreted in urine
• Deoxyuridine supression – detect vit b12 and
folate def – thymidine (radio uptake)
 NON-MEGALOBLASTIC
MACROCYTIC ANEMIA
- the RBC is round, not oval as is seen in the
megaloblastic anemia.
due to:
1. Alcoholism
2. Liver disease
3. Conditions that caused accelerated erythpoiesis
Chronic dyserythropoiteic syndrome (CDA)
- genetic predisposed
-Abnormal nuclear development of erythroid
precursor ( karyorrhexis)
- Leukocytes and platelets are not affected
Three types:
Type I – slight macrocytic anemia
Type II – normocytic anemia
Type III – normocytic or slight macrocytic
Type Anemia Binucleari NRBC NRBC Mode of Acidified
Classificati ty Multinucl Chromati Inheritan Serum
on earity n ce Test
Structur
e
I Slightly Yes No Megalobl AR Negative
macrocytic astic
II Normocytic Yes Yes Normobl AR Positive
astic
III Normocytic Occasiona Yes Normobl AD Negative
or slightly l astic
macrocytic
 FAILURE OF IRON METABOLISM
3 general etiologic process:
– Deficiency of raw material
– Defective release of stored iron
– Defective utilization of iron within erythroblast
Classified as:
1. Iron Deficiency Anemia
2. Sideroblastic Anemia
3. Anemia of Chronic Disease
 IRON DEFICIENCY ANEMIA
-Most common form of anemia especially in US.
Three causes:
– Increase physiologic demand
• Rapid growth, pregnancy, milk anemia of infancy,
excessive menstrual flow,
– Inadequate intake
• Low diet, achlorhydria, malabsorption syndrome
– Chronic blood loss
• GI blood loss, hookworm infection
 3 Stages of IDA:
1. Iron repletion – in excess supply of iron
2. Iron depletion – gradual decrease of iron supply
3. Iron deficiency
– Develops PICA and koilonychia (Spooning of the nails
– Fatigueness, dizziness, stomatitis, glossitis

59
Laboratory Diagnosis of IDA
• Hgb and HCT decrease
• MCV,MCH,MCHC = low
• Microcytic, Hypochromic
• RDW = increase
• RPI = low < 2.0
• TIBC = high
• Transferrin saturation and serum iron =low
• EPO = high
 SIDEROBLASTIC ANEMIA
- Caused by blocks in the protoporphyrin
pathway resulting defective hemoglobin and
synthesis iron overload in the
mitochondria of normoblast.
- Abnormal iron kinetics
RINGED SIDEROBLAST - excess iron accumulates
in the mitochondrial region of the immature
erythrocyte in the bone marrow and encircles
the nuclues
RINGED SIDEROBLAST
TYPES:
I. 4 aggregates of
ferritin
II. >6 aggregates of
ferritin
III. larger granule, ring
or collar around
nucleus of
normoblast
SIDEROBLASTIC ANEMIA
1. Hereditary SA
- X-linked
- δ ala synthetase defective
2. Acquired
– idiopathic
– Lead intoxication
• PBG synthase is affected
• Coproporphyrinogen oxidase defective
• ATPase – ( cation exchange)
• Blue-black deposit on gums and teeth
• Presence of basophilic stippling
• porphyrias

- Due to INH (anti –TB drug): affects vit B6 that lead

to the absence of the production of D-ALA
 ANEMIA OF CHRONIC DISEASE (ACD)
- Due to inability to use available iron for hemoglobin
production
- Impaired release of storage iron associated with
increase hepeidin levels.
Hepeidin
– liver hormone and a positive acue phase
reactant. It plays a major role in body iron regulation by
influencing intestinal iron absorption and release
storage iron from macrophages.
- inflammation and infection cause hepeidin levels
to increase; this decreases release iron from stores.
ANEMIA OF CHRONIC DISORDERS
- Second form as common cause of anemia
- infection, SLE, RA, Hodgkin lymphoma or
malignancy (neoplastic diseases)
- Release of interleukin 1 during inflammation
from macrophages, which blocks macrophage
iron release going to normoblast
 Differential Tests
Blood Serum Serum TIBC Transferrin FEP
Picture Ferritin Iron Saturation
IDA Microcytic, Low Low High Low High
Hypochrom
ic
Thalassemia - High/N High/N Norm High/N Nor
al mal
Sideroblastic Dimorphic High High Low High Mixe
Anemia d
Anemia of - High Low Low/ Low/N High
Chronic N
Inflammation
III. HEMOLYTIC DISORDER
A. Abormal of Globin Development
1. Hemoglobinopathies
2. Thalassemia
B. Increased RBC destruction
1. Hemolytic Anemia Due to Intrinsic defects
a. Membrane Defect
b. Metabolism/ Enzymopathy
2. Hemolytic Anemia Due to Extrinsic defects
A. Abormal of Globin Development
-hemoglobin disorder
-qualitative and quantitative globin synthesis
disorder
1. ) Hemoglobinopathies
– Qualitative structural abnormalities of the globin
chains
– Amino acid mutation
– Production of hemoglobin variants
2.) Thalassemia
– Quantitative abnormality in the globin chain
production
– Production maybe reduced or no production at all
Hemoglobinopathies
Classification of Hemoglobinopathies
1. ) Molecular abnormality
a.) Single amino acid substitution
b.) Two amino acid substitution
c.) Deletion
d.) Elongation
2.) Functional abnormality
a.) Aggregation
b.) Unstable hemoglobin
c.) Methemoglobin
d.) Oxygen affinity
HEMOGLOBINOPATHIES
1.) Sickle cell anemia
A.) Homozygous Hgb SS – Sickle Cell Disease
– Both β chain is affected
–α2β6Glu-Val
Complications
• Sickle Cell crises
– Vasoocclusive crises
– Infectious crises
– Splenic sequestration crises
– Aplastic crises
– Bone, joint and other crises

b.) Heterozygous Hgb AS – Sickle Cell Traits

– One β chain is affected
– α2β6Glu-Val
 Special Test
• Hgb Electrophoresis
• Dithionate solubility test
– Principle:
Red cell + saponin Hgb + Sodium dithionate Hgb S tactoids

Laboratory findings
- severe anemia ( Hgb 5-9 g/dl)
- normocytic, normochromic RBC
- aniso and poikilocytosis is present
- with leukocytosis and thrombocytosis
 Treatment
• Blood transfusion of Packed red cell
• Administration of antisickling agents
– Cyanate
– Urea
– Nitrogen mustard
– zinc procaine hydrochloride, Citiedil, and
piracetam
• Bone marrow transplantation
 Hemoglobinopathies
2.) Hgb C Disease
– Hgb CC and Hgb AC
– Amino acid substitution
at the 6th position
– Glutamic – lysine
– β chain is affected
 Other single amino acid substitution
3.) Hgb SC α2β6Glu-Valβ6Glu-Lys
4.) Hgb D α2β121Glu-Gln
5.) Hgb SD α2 β6Glu-Val β121Gln-Glu
6.) Hgb E α2β26Glu-Lys
7.) Hgb O-Arab α2 β121Glu-Lys
 Other Defect
• Hgb C- Harlem or Georgetown
– Two amino acid substitution
–α2β6Glu-Val;73Asp-An
• Hgb G-Philadelphia
– Alpha chain is affected
–α268ASn-Lys β2
• Hgb Gun Hill
– Deletion of five amino acid on β chain 91-95

• Hgb Constant Spring CS

– Elongation of the alpha chain
– Additional of 31 amino acid
– Results from the termination of the stop codon
 Unstable Hemoglobin Disease
• Precipitation and denaturation of the globin
chain producing Heinz bodies formation
• Hgb Bristol; Genova; Toronio;Koln;Seattle.etc
• Cause:
– Disrupted contact bet. Heme and globin
– Change in shape or structure of globin chain
– Alter amino acid during alpha and beta chain
interface
 Laboratory test for unstable Hgb
• Isopropanol • Heat Denaturation test
Precipitation test – Unstable Hgb are heat
– Nonpolar solvents sensitive and will
weakens the bonds of precipitate heavily when
Hgb molecule causing it incubated @ 50OC for 1
to precipitate hour
– 17% Alc + Hgb @ 37OC – Normal Hgb will show
small precipitate
– Normal Hgb : 40 min
– Unstable Hgb: 20 min
Hemoglobin Associated with Oxygen
Affinity
1.) Hgb Chesapeake
- α292Leuβ2
– Increase affinity
2.) Hgb kansas
– Decrease affinity
– α2β2102Thr
 Thalassemia
• Classification depends
on the specific globin
chain whose synthesis is
suppressed
• Types are α, β, δβ, and
γδβ
 Alpha – (α) Thalassemia
Disorder Genotype Genotypic Other
description description

Barts --/-- alpha 1/alpha α0 /α0

Hydrops 1
fetalis
Hgb H - -/ - α alpha 1/ α0 /α +
alpha 2

Hgb H/ CS - - / αCS α alpha 1/ α0 /αCS

heterozygous
CS
α -- / αα alpha 1/ α0 / α
Thalassemia -α/-α normal α+ / α+
minor alpha 1/
Prepared by: Godfrey D. Ca cca m, RM T 83
alp h a 1
 Alpha (α) - Thalassemia
Disorder Genotype Genotypic Other
Description Description
Bart’s Hydrops --/-- Alpha 1/alpha 1 αo/αo
Fetalis
Hemoglobin H --/-α Alpha 1/alpha 2 αo/α+
Disease
Hemoglobin H/ --/αCSα Alpha αo/αCS
CS 1/Heterozygous CS
α Thalassemia --/αα Alpha 1/Normal αo/α
Minor -α/-α Alpha 1/Alpha 1 α+/α+
 Disorder Characteristic Clinical findings Laboratory
findings
Barts Hydrops Hgb γ4 infants – Hgb 4-10g/dl
underweight, Blood film : marked
fetalis edematous, ascites, anisocytosis and
hepatosplenomegaly poikilocytosis,
Severe anemia hypochromia,
reticulocytosis, and
increase nRBC

Hgb H Hgb β4 Mental retardation Hgb 8.9 – 12 g/dl

Hepatoslenomegaly Reticulocytosis
moderate anemia Anisocytosis and
poikilocytes

Hgb H/ CS Compose of two Same with Hgb H Same with Hgb H

normal beta chain,
one normal alpha
chain and one
abnormal alpha with
171 a.a.

α Thalassemia Losing at least one None to Mild anemia Hgb and Hct are
alpha chain usually at the lower
minor limit
poikilocytosis
Bart’s Hydrops fetalis BS
 β Thalassemia
• Four different classification:
– Thalassemia Major ( Cooley’s Anemia )
• Β0 / β0
• β + / β+
• β0 / β+
• δβ Lepore ( Hgb Lepore )
– Thalassemia Intermedia
– Thalassemia Minor
– Thalassemia Minima
 β Thalassemia Major (Cooley’s
Anemia)
• Clinical Findings
• Severe anemia within 1st
year of life
• Skeletal deformities with
frontal bossing, cheek bone
and jaw protussion
• Hepatosplenomegaly
• Cardiomegaly
• Hyperslenism
• Chronic leg ulcers
• Gallstone formation
• Retarded growth and sexual
development
• Laboratory findings
• • Hg 2.5 -6.5 g/dl
• Numerous nRBC
• Marked aniso
and
poikilocytosis
• RPI less than 3
• Increase Hgb F
• concentration
• Serum ferritin increase
and iron overload
β Thalassemia Major (Cooley’s
Anemia)
 Hgb Lepore

• Compose of two normal alpha chain and two

abnormal non-alpha chains (fusion of N-
terminal of delta chain and C-terminal of beta
chain)
• Crossing over of the delta and beta genes
resulted to lack of intact delta and beta gene
B.) Increased RBC destruction
1. Hemolytic Anemia Due to Intrinsic defects
a. Membrane Defect
b. Metabolism/ Enzymopathy
2. Hemolytic Anemia Due to Extrinsic defects
 CLASSIFICATION OF HEMOLYTIC ANEMIA
INTRINSIC HEMOLYTIC ANEMIA
1.) HEREDITARY
a.) MEMBRANE DEFECTS
Hereditary Spherocytosis Hereditary
Elliptocytosis Hereditary Pyropoikilocytosis
Hereditary Stomatocytosis
b.) Enzyme defects
G6PD deficiency
Pyruvate kinase deficiency Glutathione
reductase deficiency
c.) Hemoglobinopathies

2.) ACQUIRED
Paroxysmal Nocturnal Hemoglobinuria
EXTRINSIC HEMOLYTIC ANEMIA
1.) HEREDITARY
LCAT deficiency
Abetalipoproteinemia

2.) ACQUIRED
Immune mediated
Mechanical, thermal, chemical damage
Infectious agents
Hypersplenism
Secondary to liver or renal disease
Secondary to hypertension
 INTRINSIC HEMOLYTIC ANEMIA
A. MEMBRANE DEFECT
1. Hereditary Spherocytosis
– Decreased surface area: volume ratio
– Limited capacity to expand in hypotonic solution
– Defective binding of spectrin to protein 4.1
– Spectrin deficiency
– Complication: Aplastic crisis (viral)
– SPLENOMEGALY
– INCREASED OSMOTIC FRAGILITY WITH MCHC
INCREASED
2. Hereditary elliptocytosis
– One-fifth as common as HS
– Large number of elliptocytes on the blood film
– Deficiency of protein 4.1(spherocytic HE)
– Spectrin dimer-dimer interaction and a defective
ankyrin-protein 3 interaction
– SPLENOMEGALY

Spherocytic Hereditary Elliptocytosis

-This subgroup accounts for 10% of cases.
-A mild to moderate hemolytic anemia and
splenomegaly are present, with both elliptocytes and
spherocytes,
- abnormal osmotic fragility and autohemolysis tests.
- Poikilocytes and fragments are usually absent.
- The molecular basis of this subtype is unknown.
3. Hereditary Stomatocytosis (Hydrocytosis)
and Hereditary Xerocytosis
–Heterogenous group of red cell membrane disorders
– Increased volume and a decreased surface-to-volume
ratio (Hydrocytosis)
– Increased surface-to-volume ratio-with target cells
(Xerocytosis)
– Mild to moderate hemolytic anemia
–Altered membrane permeability to cations
Laboratory findings:
Stomatocytosis – increase in osmotic fragility and
autohemolysis
Xerocytosis – decrease osmotic fragility
4. Hereditary pyropoikilocytosis
– Extremely rare
(HPP)
– Extreme anisocytosis and micropoikilocytosis with
budding red cells, fragments, spherocytes,
elliptocytes and other bizarre shaped cells
– Spectrin dimer-dimer association
– RBC with increased Calcium
– show budding and fragmentation when heated to
49°C, HPP red cells fragment at 45–46°C.
5. Southeast Asian Ovalocytosis
- This subgroup occurs with high frequencies
(20-30%) in certain populations of the Far East,
particularly Malaysia.
- Hemolysis is usually absent or mild.
- The erythrocytes are less elongated and some
have the appearance of stomatocytic ovalocytes.
- Many cells contain one or two transverse
ridges or a longitudinal slit.
- This condition is associated with increased
resistance to malaria.
- The underlying defect is related to a mutation
in band 3
6. Paroxysmal Nocturnal Hemoglobinuria
-an acquired clonal stem cell disorder characterized
by the production of abnormal erythrocytes,
granulocytes, and platelets .
– Cells become sensitive to complement when slight
lowering of blood pH occurs
– defects in molecular structure of the Decay Accelerating
Factor
Laboratory Testing:
a.) Sugar Water Screening test/ Sucrose Hemolysis Test
• The red cell lyses after treatment with LISS sugar
solution which is incubated @37 ° C
b.)HAM’s Acidified serum test
• RBC in PNH is sensitive after acidification of serum
(0.2N HCl)
 B. METABOLIC DEFECT/ ENZYMOPATHY
1.) Methemoglobin reductase deficiency
– Formation of Ferric iron
– Inherited or Acquired
– Methylene blue treatment

2.) G-6-PD defeciency

– Most common enzymopathy
– NADPH in the RBC is linked to glutathione reduction
– Formation of Heinz bodies
– Episodic hemolysis induced by drugs and other
oxidant
– Severe, chronic non-spherocytic hemolytic anemia
– Favism
G-6-PD deficiency
Genetics
• Sex-linked
• Males affected
• Females – carrier

Diagnosis
• Screening tests
• Direct enzyme assay on red cells
• During hemolytic blood film may show
– Contracted and fragmented cells
– ‘bite’ cells and ‘blister’ cells
– Heinz bodies (supravital stain)
Ascorbate Cyanide Test
• When blood is incubated with a solution of sodium cyanide and
sodium ascorbate, hydrogen peroxide is generated from the
coupled oxidation of ascorbate and hemoglobin. Cyanide inhibits
catalase, hydrogen peroxide is available to oxidize hemoglobin, and
the brown color of methemoglobin is discernible. This occurs more
rapidly in G6PD-deficient cells than in normal cells.

Fluorescent Spot Test

• Whole blood is added to a mixture of glucose-6-phosphate (G6P),
NADP, saponin, and buffer, and a spot of this mixture is placed on
filter paper and observed for fluorescence with ultraviolet light. If
G6PD is present, NADP is converted to NADPH. Because
phosphogluconate dehydrogenase is present in most hemolysates,
further NADP is converted to NADPH. NADPH fluoresces but NADP
does not.

Quantitative Assay of G6PD

• For G6PD, most assays are based on the rate of reduction of NADP
to NADPH, measured spectrometrically at 340 nm, when a
hemolysate is incubated with G6P.
Peripheral smear – G6PD deficiency
 Treatment
• Avoidance of offending agents
• Folic acid supplementation
 3. Pyruvate kinase deficiency
– Most common enzyme deficiency of the EM pathway (90%
of energy)
– Marked reduction in ATP
–K and H20 are lost cell shrinkage, distortion, spiculation
Diagnosis:
Fluorescent Spot Test
Pyruvate kinase catalyzes the phosphorylation of ADP to
ATP by phosphoenolpyruvate (PEP) with the formation of
pyruvate. Pyruvate then reduces any NADH present to NAD
with the formation of lactate. Loss of fluorescence of
NADH under ultraviolet light is observed as evidence of the
presence of PK.
EXTRINSIC HEMOLYTIC ANEMIA
1.) Physical agent
– Heat, trauma, microangiopathic HA ( HUS – E. coli, TTP)
– DIC

2.) Infectious agent

– Malaria, Oroya fever,IM, Mycoplasma pneumoniae,
Clostridium perfringens

3.)Immune hemolytic anemia

– AIHA (cold and warm)
– Isoimmune HA (Rh and ABO)
– Drug induce HA ( penicillin, cephalosphorin, methyldopa)
– PCH ( Donath-Landsteiner Ab, anti-P)
1. Physical agent
a.)HEAT
- Extensive burns produce hemolytic
anemia, probably because of direct damage
to red cells.
- Gross hemoglobinuria may occur in
patients with severe burns.
- The blood film may show remarkable
morphologic abnormalities of the red cells,
including budding fragmentation of the
membrane and microspherocytosis.
b.) Traumatic Hemolysis
- Hemolytic anemia characterized by striking
morphologic abnormalities of the red cells, which
include fragments (schistocytes) and irregularly
contracted cells (triangular cells, helmet cells),
has been attributed to physical trauma to the red
cells.
c.) External mechanical trauma
March hemoglobinuria, a condition that is
seen in soldiers after a long march and in joggers
and other athletes following long practices,
particularly on hard surfaces.
d.) Thrombotic Microangiopathy (Microangiopathic
Hemolytic Anemia)
- microvascular occlusive disorders characterized
by systemic or intrarenal aggregation of platelets,
thrombocytopenia, and mechanical injury to
erythrocytes.
i.) Hemolytic Uremic Syndrome (HUS)
- occurs most commonly in infants less than 2 years
of age and is often preceded by an infection associated
with diarrhea.
- It is likely that some of these cases are due to one
of several serotypes of Escherichia coli that produce a
verotoxin (named for its toxicity against African green
monkey kidney [vero] cells).
- clots form causing renal damage
ii.) Thrombotic Thrombocytopenic Purpura
- may occur at any age but has a peak incidence in the
third decade
- it occurs more often in females than in males.
- deficiency in enzyme ADAMTS13 that is responsible for
breaking down large von Willebrand factor multimer.
- when multimers are not broken down, clots form,
causing RBC fragmentation and CNS impairment.

iii.) Disseminated Intravascular Coagulation (DIC)

- systemic clotting is initiated by activation of coagulation
cascade due to toxins or conditions that trigger release of
procoagulant
- fibrin is deposited in small vessel causing RBC
fragmentation
iv.) Pre-eclampsia/Eclampsia
- are microangiopathic disorders occurring
with pregnancy and sharing some features of
HUS or TTP.
- Approximately 4-39% of patients with
severe pre-eclampsia develop HELLP
syndrome:
H emolysis,
E levated L iver enzymes and
L ow P latelet count.