NORMOCYTIC, NORMOCHROMIC ANEMIA CLINICAL AND LAB FINDINGS IN H.S.

HEMOLYTIC ANEMIA 3 KEY CLINICAL MANIFESTATIONS:

 May be acquired or hereditary abnormality in the basic  ANEMIA

membrane structure, erythocytic enzyme or hemoglobin  JAUNDICE
sturcture.  SPLENOMEGALY
 All are normocytic, normochromic anemia, with
reticulocytosis ADDITIONAL TESTS

HEMOLYTIC ANEMIAS OSMOTIC FRAGILITY TEST:

CATEGORIES OF HEMOLYTIC ANEMIA  demonstrates increased RBC fragility in blood specimens

INTRINSIC DEFECT (Hereditary)

• Membrane Defect
• Enzyme Defect
• Hemoglobin Defect (Hemoglobinopathies)

EXTRINSIC DEFECT

• Immune
• Non-Immune

HEMOLYSIS BY MEMBRANE DEFECT

• Hereditary Spherocytosis
• Hereditary Elliptocytosis
• Hereditary Stomatocytosis
• Hereditary Acanthocytosis
• Paroxysmal Nocturnal Hemoglobinuria
HEREDITARY RBC MEMBRANE ABNORMALITIES
 MUTATIONS THAT ALTER MEMBRANE STRUCTURE
o HEREDITARY SPHEROCYTOSIS
 Heterogeneous group of hemolytic anemias caused by
defects in proteins that disrupt the vertical interactions
between transmembrane proteins and the underlying
protein cytoskeleton
 Most common membrane defect
 Autosomal dominant
 Characterized by splenomegaly, variable degree of
anemia, and spherocytes
 Increased permeability of the membrane to sodium
 RBCs acquire a decreased surface area-to-volume ratio,
and the cells become spherical
INTRINSIC DEFECT: HEREDITARY SPHEROCYTOSIS
 MOST COMMON MEMBRANE DEFECT
 Autosomal dominant
 Splenomegaly and spherocytes in the PBS
 Increased permeability to Sodium
 Decreased surface area to volume ratio
 Increased Osmotic fragility and Bilirubin, MCHC
 OFT is a sensitive test
in which the RBCs have decreased surface area-to-
volume ratio
 Blood is added to a series of tubes with increasingly
hypotonic NaCl solutions
 Standard volume of fresh heparinized blood is mixed with
NaCl solution ranging from 0.85% to 0.0%
 Normal RBCs: initial hemolysis at 0.45% NaCl complete
hemolysis occurs between 0.35% and 0.30% NaCl
 EOSIN-5’-MALEIMIDE (EMA) BINDING TEST
o More sensitive alternative test for confirmation of HS
o Suitable for low-volume pediatric specimens
o Can be performed within 3 hours
o Specimens are acceptable for analysis up to 7 days
after collection
 SDS-PAGE
 ACIDIFIED GLYCEROL LYSIS TEST
 AUTOHEMOLYSIS TEST
 HYPERTONIC CRYOHEMOLYSIS TEST
HEREDITARY ELLIPTOCYTOSIS
o Heterogeneous group of hemolytic anemias caused by defects
in proteins that disrupt the horizontal or lateral interactions in
the protein cytoskeleton
o Disease is more common in Africa and Mediterranean regions,
where there is high prevalence of malaria
o RBCs are biconcave and discoid at first, but become elliptical
over time after repeated exposure to the shear stress in the
peripheral circulation
o Autosomal dominant; most persons are asymptomatic due to
normal RBC life span
o Membrane defect is caused by polarization of cholesterol at
the ends of the cell rather than around the pallor area
o There is membrane loss and decreased surface area-to-
volume ratio
INTRINSIC DEFECT: HEREDITARY ELLIPTOCYTOSIS
o Autosomal dominant; asymptomatic
o >25% Ovalocytes
o Caused by polarized cholesterol at the ends of the cell
instead around the pallor area
o HEREDITARY PYROPOIKILOCYTOSIS – subtype of HE
seen in black ppl.
HEREDITARY PYROPOIKILOCYTOSIS
o Severe form of HE that exists in either the homozygous or
compound heterozygous state
o PBS shows extreme poikilocytosis with fragmentation,
microspherocytosis, and elliptocytes which shows marked
thermal sensitivity
TREATMENT
o Asymptomatic: NO TREATMENT
o Symptomatic: SPLENECTOMY
HEREDITARY OVALOCYTOSIS (SOUTHEAST ASIAN
OVALOCYTOSIS)
o Condition caused by mutation in the gene for band 3 that
results in increased rigidity of the membrane and
resistance to invasion by malaria
o Autosomal dominant and all patients are heterozygous
LAB FINDINGS:
 Oval RBCs (30% of the RBCs)
HEREDITARY OVALOCYTOSIS (SOUTHEAST ASIAN
OVALOCYTOSIS)
 Condition caused by mutation in the gene for band 3 that
results in increased rigidity of the membrane and
resistance to invasion by malaria
 Autosomal dominant and all patients are heterozygous
LAB FINDINGS:
 Oval RBCs (30% of the RBCs)
MUTATION THAT ALTER MEMBRANE TRANSPORT
PROTEINS
HEREDITARY STOMATOCYTOSIS
 Group of heterogeneous conditions in which the RBC
membrane leaks monovalent cations (particularly sodium)
 Autosomal dominant
 With variable degree of anemia
 Up to 50% stomatocytes on the blood smear
INTRINSIC DEFECT: HEREDITARY STOMATOCYTOSIS
 Autosomal dominant: >50% Stomatocytes
 Abnormal Sodium and Potassium permeability.
 Causes RBC Swelling
MAJOR CATEGORIES OF HEREDITARY
STOMATOCYTOSIS
OVERYDRATED HEREDITARY STOMATOCYTOSIS
 Very rare hemolytic anemia due to a defect in the
membrane cation permeability that causes the RBCs to be
overhydrated
 Autosomal dominant
 RBC membrane is excessively permeable to sodium and
potassium at 37C
 Diagnostic feature: 5-50% stomatocytes on PBS,
macrocytes (MCV:110-150fL), decreased MCHC (24-
30g/dL), reticulocytosis, reduced erythrocyte potassium
and increased erythrocyte sodium concentration
DEHYDRATED HEREDITARY STOMATOCYTOSIS/
HEREDITARY XEROCYTOSIS
 Autosomal dominant
 due to a defect in membrane cation permeability that
causes the RBCs to be dehydrated
 Most common form of stomatocytosis
 Due to mutations in the PIEZO1 gene
 Diagnostic feature: Mild to moderate anemia,
reticulocytosis, jaundice, mild to moderate splenomegaly,
fetal loss, hydrops fetalis, and neonatal hepatitis
 Stomatocytes (fewer than 10%), target cells, burr cells are
also seen
INTRINSIC DEFECT: HEREDITARY XEROCYTOSIS
 Permeability disorder: thermal instability of spectrin in vitro
 Crenated cell due to increased Na intake of the cell
 Increased MCHC
OTHER MEMBRANE DEFECTS WITH STOMATOCYTES
 FAMILIAL PSEUDOHYPERKALEMIA
o Rare disorder in which excessive potassium leaks out of
the RBCs at room temperature in vitro but not at body
temperature in vivo
o Additional stomatocytes may be observed on PBS
 CRYOHYDROCYTOSIS
o Mild to moderate hemolytic anemia with leakage of
sodium and potassium from the RBCs; also with
stomatocytosis
o RBCs have marked swelling and hemolyzes when
stored at 4C for 24-48 hours
 Rh DEFICIENCY SYNDROME
o Rare hereditary condition in which the expression of Rh
membrane proteins is absent or decreased
o Patients are characterized with mild to moderate
hemolytic anemia, stomatocytes and occasional
spherocytes may be observed
INTRINSIC DEFECT: HEREDITARY ACANTHOCYTOSIS
 Associated with steatorrhea, neurological and retinal
abnormalities
 50 to 100% acanthocytes
 Increased Cell Lecithin and Cholesterol ratio
 Abetalipoproteinemia
 Normal blood indices
MEMBRANE DEFECTS WITH ACANTHOCYTES
 NEUROACANTHOCYTOSIS
 Group of rare inherited disorders characterized by
neurologic impairment and acanthocytes on the
peripheral blood
o ABETALIPOPROTEINEMIA
 Rare autosomal recessive disorder characterized by
fat malabsorption, progressive ataxia, retinitis
pigmentosa, and acanthocytosis
 Caused by mutations in the MTP gene
 50-90% of the RBCs are acanthocytes
 Treatment: high doses of Vit. A and E
 MCLEOD SYNDROME (MLS)
 X-linked disorder caused by mutations in the KX gene
 Patients have variable acanthocytosis, mild anemia,
and late-onset and slowly-progressive chorea,
peripheral neuropathy, myopathy, and
neuropsychiatric manifestations
 CHOREA ACANTHOCYTOSIS
 Rare autosomal recessive disorder
 Characterized by chorea, hyperkinesia, cognitive
impairments, and neuropsychiatric symptoms
 5-50% of RBCs on the PBS are acanthocytes
 Caused by mutations in the VPS13A gene
ACQUIRED RBC MEMBRANE ABNORMALITIES
ACQUIRED STOMATOCYTOSIS -occurs frequently as
drying artifact on Wright-stained peripheral blood films
SPUR CELL ANEMIA -severe liver disease develop a
hemolytic anemia with acanthocytosis
NOTE: In normal individuals, 3-5% of RBCs may be
stomatocytes
PAROXYSMAL NOCTURNAL HEMOGLOBINURIA
 Rare chronic intravascular hemolytic anemia caused by an
acquired clonal hematopoietic stem cell mutation that
results in circulating blood cells that lack CD55 and CD59
 Acquired membrane defect in which the red cell
membrane has an increased sensitivity for complement
binding as compared to normal erythrocytes
 ALL CELLS are abnormally sensitive to lysis by
complement
 Characterized by: pancytopenia, chronic intravascular
hemolysis causing hemoglobinuria and hemosiderinuria at
an acid pH at night
 It is noted for low LAP score
 HAM and sugar water hemolysis test/sucrose hemolysis
test: traditional test for PNH diagnosis
 Standard test: FLOW CYTOMETRY to detect deficiencies
of GPI-linked proteins (CD55 and CD59)
NOTE:
 TREATMENT: ECULIZUMAB (SOLIRIS) for treatment of
hemolysis in PNH
 :humanized monoclonal antibody that binds to
complement C5, prevents cleavage to C5a and C5b, and
thus inhibits the formation of MAC
 treatment of choice for px with classic PNH
 px taking this drug have increased risk of infections with N.
meningitides
 :before eculizumab was formulated, the major cause of
death is THROMBOSIS
HEMOGLOBINOPATHIES
(Qualitative Hemoglobin Defect)
 HEMOGLOBINOPATHY: refers to a disease state
involving the hemoglobin molecule; structural defects in
hemoglobin
 Group of inherited disorders causing structurally abnormal
globin chain synthesis due to amino acid substitutions;
changes in RBC deformability and electrophoretic mobility
 MOST COMMON POIKILOCYTE SEEN: TARGET CELLS
 All hemoglobinopathies result from genetic mutation in one
or more genes that affect hemoglobin synthesis
 All affect hemoglobin synthesis in one of two ways:
Qualitatively or Quantitatively
2 CATEGORIES
QUALITATIVE HEMOGLOBINOPATHIES
 Hemoglobin synthesis occurs at a normal or near-normal
rate, but the hemoglobin molecule has an altered amino
acid sequence within the globin chains
 Structural defects
QUANTITATIVE HEMOGLOBINOPATHIES
 • Result in reduced hemoglobin synthesis but do not
affect the amino acid sequence of the globin chains
-ex: THALASSEMIA
SICKLE CELL DISEASE (Hb SS)
 CAUSE: when VALINE replaces GLUTAMIC ACID at 6 th
position on both beta chains
 Mutation occurs in nucleotide 17
 Results in decrease hemoglobin solubility and function
 Defect is inherited from both parents
 Occurs most commonly in African-American, African,
Mediterranean, Middle East, and Indian populations
 No Hgb A is produced (80% Hgb S and 20% Hgb F); Hgb
A2 is variable
 Hemoglobin crystallizes inside erythrocytes giving it a
classic sickled-shape of RBCs
 MOST COMMON FORM OF HEMOGLOBINOPATHY
NOTE:
 Hgb F: is the compensatory hemoglobin (1-20%)
 Hgb A2: normal or slightly increased (2-5%)
 SICKLE CELL ANEMIA: was first reported by HERRICK in
1910 in a West Indian student with severe anemia
 Highest prevalence occurs in Africans, Indians experience
much milder form
 In 1927: Hahn and Gillespie described the pathologic
basis of the disorder and its relationship to the hemoglobin
molecule
CLINICAL FINDINGS
 RBCs become rigid and trapped in capillaries; blood flow
restriction causes lack of oxygen to tissues resulting in
TISSUE NECROSIS
 All organs are affected; hyposplenism and joint swelling
also occur
 Vaso-occlusive crisis occurs with increased bone marrow
response to hemolytic anemia
 Apparent immunity to Plasmodium falciparum
 RBCs containing Hgb S become susceptible to
hemolysis, and progressive hemolytic anemia and
splenomegaly become evident
 Diagnosis is made after 6 months of age, with life
expectancy of 50 years with proper treatment
 ACUTE CHEST SYNDROME: leading cause of death
among adults with SCD
 BACTERIAL INFECTION: frequent cause of death of
children <3 year old with SCD
NOTE:
• AVASCULAR NECROSIS: impaired blood supply to the
head of femur and humerus; develop by 35 year of age
• KIDNEY FAILURE is the most common outcome
• VASO-OCCLUSIVE CRISIS: can be initiated by
SURGERY, TRAUMA, PREGNANCY and HIGH
ALTITUDES
• VASO-OCCLUSIVE CRISIS: hallmark of SICKLE CELL
DISEASE
• AUTOSPLENECTOMY: gradual loss of splenic function;
evidence by the presence of HJ bodies and Pappenheimer
bodies
• ACUTE CHEST SYNDROME: fever, chest pain and
presence of pulmonary infiltrates
• BACTERIAL INFECTION: life-threatening infection with S.
aureus, S. pneumoniae, H. influenza
• BOSENTAN: txt of choice for pulmonary hypertension
• MOST COMMON CAUSE DEATH: INFECTION/SEPSIS
and CONGESTIVE HEART FAILURE
2 FORMS OF SICKLE CELLS
REVERSIBLE SICKLE CELLS
• Hgb S-containing erythrocytes that change shape in
response to oxygen tension
• Circulate as normal biconcave-disc RBCs when fully
oxygenated but undergo hemoglobin polymerization in
cases of decrease oxygen conditions
IRREVERSIBLE SICKLE CELLS
• Do not change their shape regardless of the change in
oxygen tension or degree of hemoglobin polymerization
• Seen in peripheral blood as elongated sickle cells with a
point at each end
• Recognized as abnormal by the spleen and removed from
the circulation
LABORATORY DIAGNOSIS
• Severe NORMOCYTIC/NORMOCHROMIC hemolytic
anemia with polychromasia
• Premature release of reticulocytes
• BM erythroid hyperplasia
• (+) Sickle cells, target cells, nRBCs, Pappenheimer
bodies, and HJ bodies
• Increased bilirubin and decreased haptoglobin
TEST FOR HEMOGLOBIN S DETECTION
SODIUM METABISULFITE TEST
• Older screening test that detects Hgb S insolubility by
inducing sickle cell formation on a glass slide
• Drop of blood is mixed with a drop of 2% sodium
metabisulfite (reducing agent) on a slide, and the mixture
is sealed under a coverslip
• (+) HOLLY-LEAF APPEARANCE OF ERYTHROCYTES
HEMOGLOBIN SOLUBILITY TEST
• Most common screening test for Hgb S
• Blood is added to a buffered salt solution containing a
reducing agent such as sodium hydrosulfite/sodium
dithionite, and a detergent-based lysing agent called
saponin
INTERPRETATION:
• (+): TURBID • SICKLING OF RBCS CAN OCCUR DURING EXTREME
• (-): CLEARNOTE: EXERTION AND DECREASE OXYGEN TENSION
• (+) HEMOGLOBIN SOLUBILITY SCREENING TEST
NOTE: • WITH APPARENT IMMUNITY TO Plasmodium falciparum
• SAPONIN: dissolves membrane lipids causing the release
of hemoglobin from the RBCs HEMOGLOBIN C DISEASE (Hgb CC)
• DITHIONITE: reduces the iron from ferrous to ferric form • 2nd most common hemoglobinopathy in U.S
• Milder disease as compared to Hgb S
HEMOGLOBIN ELECTROPHORESIS • CAUSE: LYSINE replaces GLUTAMIC ACID at 6th position
on both beta chains
• Common first step in the confirmation of • Defect is inherited from both parent
hemoglobinopathies • Occurs in the African-American and African populations
• No Hgb A is produced; approximately 90% Hgb C, 2%
• Hgb S migrates with Hgb D and Hgb G in an alkaline
Hgb A2 and 7% Hgb F
electrophoresis; and can be confirmed by performing
• Hgb C polymerizes under low oxygen tension and forms a
CITRATE AGAR ELECTROPHORESIS
short, thick crystal within the RBCs after polymerization
TREATMENT • With less splenic sequestration and hemolysis

• NEONATAL SCREENING LABORATORY DIAGNOSIS

• PROPHYLACTIC PENICILLIN THERAPY
• NORMOCYTIC/NORMOCHROMIC ANEMIA with target
• BM TRANSPLANT
cells; characterized by intracellular rod-like Hgb C crystals
• HYDROXYUREA
• With INCREASED reticulocyte count and nRBCs
• ADEQUATE HYDRATION
• (-) Hemoglobin solubility test
• ANALGESICS
• Hgb C migrates with Hgb A2, E, and O in alkaline
• AVOIDANCE OF LOW-OXYGEN ENVIRONMENTS
electrophoresis
• AGGRESSIVE ANTIBIOTIC THERAPY
• MORPHINE HEMOGLOBIN C TRAIT (Hgb AC)
• BLOOD EXCHANGE TRANSFUSION
• COMPOSED OF:
NOTE: o 60% Hgb A
o 30% Hgb C
• Prophylactic penicillin V: 125 mg (2x a day):
recommended for 3 months to 3 years of age; 250 mg (2x
HEMOGLOBIN SC DISEASE (Hgb SC)
a day): 3-5 year old
• HYDROXYUREA: relieve sickling disorder by increasing • Double heterozygous condition where there an abnormal
Hgb F levels sickle gene from one parent and an abnormal C gene from
• ADEQUATE HYDRATION: maintains good flow of blood the other parent is inherited
and prevents vaso-occlusive crisis • Most common compound heterozygous syndrome that
• ANALGESICS: for pain management (paracetamol/ results in a structural defect in the hemoglobin molecule
acetaminophen) • At 6th position, GLUTAMIC ACID is replaced by VALINE
• MORPHINE: acute VOC attacks and by LYSINE on the other B-globin chain
• BLOOD EXCHANGE TRANSFUSION: txt of choice for • Seen in African, Mediterranean, and Middle Eastern
severe VOC and acute chest syndrome populations
• Resembles mild form of SCD
SICKLE CELL TRAIT (HEMOGLOBIN AS)
NOTE: 50% hgb S and 50% hgb C
• Heterozygous and benign condition
• Generally asymptomatic and present with no significant LAB FINDINGS
clinical or hematologic manifestations
• CAUSE: when VALINE replaces GLUTAMIC ACID at 6th • Moderate to severe NORMOCYTIC/NORMOCHROMIC
position on one beta chain anemia with TARGET CELLS
• Defect is inherited from one parent; one normal beta chain
can produce some Hgb A • (+) Hgb SC crystals
• Approximately 60% Hgb A and 40% Hgb S are produced,
with normal amounts of Hgb F and A2 • SICKLE CELLS and C crystals (rarely)
• Most common hemoglobinopathy in the United States
(+) Hemoglobin solubility tes
CLINICAL FINDINGS
HEMOGLOBIN E DISEASE (Hgb E)
• NORMOCYTIC/NORMOCHROMIC ANEMIA (RARE)
• CAUSE: when LYSINE replaces GLUTAMIC ACID at 26th
position on beta chain
• Found more commonly in African, Mediterranean,
Middle Eastern, and Asian populations (Thailand)
• 3rd most common cause of anemia in US
• Symptoms less severe than sickle cell anemia but more
severe than Hgb C disease
• HOMOZYGOUS: mild anemia with macrocytes and target
cells
• HETEROZYGOUS: asymptomatic
• Hemoglobin E migrates with hemoglobin A2, C, and O in
an alkaline electrophoresis
• When hemoglobin E is combined with B-thalassemia, the
disease becomes more severe and closely resembles B-
thalassemia major, requiring regular blood transfusions
NOTE: FIRST DESCRIBED IN 1954
BOTH QUANTI (DECREASED PRODUCTION OF GLOBIN
CHAIN) AND QUALI (AA SUBSTITUTION IN THE GLOBIN
CHAIN) DEFECT
LAB FINDINGS:
• (-) HEMOGLOBIN SOLUBILITY TEST
• HOMOZYGOUS STATE: >90% Hgb E, very low MCV (55-
65fL), few to many target cells, and a normal reticulocyte
count
• HETEROZYGOUS STATE: mean MCV of 65fL, slight
erythrocytosis, target cells, and approximately 30%-40%
Hgb E
• Hgb D-Punjab and Hgb D-Los Angeles: glutamine is
substituted for glutamic acid at 121st position in the B chain
• Hgb G-Philadelphia: substitution of asparagine by lysine
at position 68
• Hgb G-Philadelphia: most common G variant
encountered in African Americans
• Hgb D and G do not sickle and yield a negative
hemoglobin solubility test result
NOTE:
• Hgb D-Punjab: 3% of the population in Northwestern India
• Hgb D-Los Angeles: <2% of African Americans
HEMOGLOBIN S-B-THALASSEMIA
• Most common cause of sickle cell syndrome in patients of
Mediterranean descent
• Second to Hb SC disease among all compound
heterozygous sickle disorders
Hemoglobin M
• Affects alpha, beta, or gamma chain
• Histidine replacement of Tyrosine
• Increased oxidation of Iron
• Methemoglobinemia

OTHER HEMOGLOBINOPATHIES

HEMOGLOBIN C-HARLEM (HEMOGLOBIN C-

GEORGETOWN)

• Has double substitution on the B chain (substitution of

valine for glutamic acid at 6th position of the B chain and
substitution at 73rd position of aspartic acid for asparagine
• (+) solubility test result
• On cellulose acetate: Hgb C-Harlem migrates in the C
position
• On citrate agar electrophoresis: Hgb C-Harlem migrates in
the S position

HEMOGLOBIN O-ARAB

• Is a B chain variant caused by substitution of lysine for

glutamic acid at the 121st amino acid position
• Rare disorder found in Kenya, Israel, Egypt, Bulgaria,
and African Americans
• HOMOZYGOUS: mild hemolytic anemia, with many target
cells , (-) hemoglobin solubility test and splenomegaly
• On cellulose acetate: it migrates with Hgb A2, C, and E
• The only hemoglobin to move just slightly away from the
point of application toward the cathode on citrate agar at
an acid pH

HEMOGLOBIN D AND G