[TRANS] LECTURE UNIT 05: HEMOLYTIC ANEMIA (INTRINSIC DEFECTS)

INTRODUCTION stercobilin, which will give the normal brown color

of the stool.
• The formation and destruction of RBCs normally occurs
extravascularly, it could either be in the spleen or in the
liver.

• (1) RBCs have a lifespan of 120 days inside the blood

vessels.
o When they serve their purpose, they will become
senescent or old.
o They will die via apoptosis or phagocytosis.
• (2) Heme and globin portions will be released inside the
phagocyte.
• (3) Globin portion will be recycled to form the amino
acids and will be reused for further protein synthesis.
• (4) Heme portion will release iron (Fe3+) which will be
carried in the blood by transferrin (iron transport protein)
o (4.1) Iron-transferrin complex will go to the liver and
will become ferritin.
o (4.2) Ferritin will be processed by the liver and will
be released by the liver as iron bound to transferrin.
o (4.3) Iron and transferrin will go to the iron deposits OVERVIEW OF HEMOLYTIC ANEMIA
in the bones
o (4.4) Iron in the bones will be reused for another
process of RBC formation.
• (5) Heme portion will release biliverdin will be
converted to unconjugated bilirubin
o (5.1) Unconjugated bilirubin, together with albumin,
will be transported to the liver.
▪ In the liver, conjugation of bilirubin happens.
o (5.2) Conjugated bilirubin will go into the small
intestine.
o (5.3) In the small intestine, bilirubin will be acted
upon by the bacteria, converting it into urobilinogen
(a colorless substance). • Intracorpuscular – within the RBC
▪ Urobilinogen can be filtered out through the o Hereditary
kidneys or through the feces.
▪ Membrane defects (Hereditary spherocytosis,
o (5.4) In the kidney, urobilinogen will be converted hereditary elliptocytosis, hereditary
into urobilin that will contribute to the yellow color of pyropoikilocytosis, hereditary stomatocytosis)
the urine. ▪ Enzyme defects (G6PD)
o (5.4) In the large intestine, urobilinogen will be ▪ Hemoglobin defects (hemoglobinopathies, sickle
converted to a brown-colored substance, cell disorders, thalassemias)

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 LECTURE UNIT 05: HEMOLYTIC ANEMIA (INTRINSIC DEFECTS)

o Acquired Intravascular Hemolysis

▪ Membrane defects
▪ Paroxysmal Nocturnal Hemoglobinuria (PNH)
• Extracorpuscular – outside the RBC
o Auto-immune Albumin

▪ Warm antibody Haptoglobin Hemopexin

▪ Cold antibody
▪ Transfusion reactions
▪ Drug-associated
o Non-immune
▪ Hypersplenism
▪ Infections (malaria)
▪ Mechanical trauma to RBCs
▪ Liver disease (Spur cell)
Hemolytic Anemia

• Mechanism: primarily to increased RBC destruction

o There is increased rate of destruction wherein the
rate of destruction exceeds the bone marrow’s
capacity to produce RBCs
o If there is increased destruction, then there would be
• If there is an increase in activation of complement
hemolytic anemia
system, presence of antibodies, or other protozoan
• Effect: shortened RBC survival infections, it would cause cell lysis.
o Typical lifespan of RBC: 120 days o When RBCs lyse, they release hemoglobin
• Defect: • For hemoglobin dimers, hemoglobin will attach to
haptoglobin.
o Intrinsic: defect on the RBC itself
o When haptoglobin-hemoglobin complexes are
▪ Usually hereditary, could either be membrane present in the blood stream, they will go to the
defect, metabolic defect or hemoglobin defect hepatocyte, wherein they will be converted into
▪ Corrected by transfusion bilirubin → urobilinogen → stercobilin.
o Extrinsic: due to factors Outside the RBC acting
• However, in cases of excessive intravascular
upon it
hemolysis, the haptoglobin which normally attaches to
▪ Could either be from abnormal environmental Hgb, and the hemopexin which normally attaches to
factors that damage the normal RBC such as: the heme portion will be exhausted.
 increased sensitivity to complement system, o The amount of haptoglobin and hemopexin are
 presence of coating antibodies (lysis of RBCs) inadequate.
 substances in the plasma/conditions affecting o Thus, the excess amount of hemoglobin will be
the anatomy of the circulatory system filtered out through the kidneys, causing the typical
(presence of defective valves) manifestations of intravascular hemolysis, such as:
 infections (malarial or other infectious agents
in which they reside the RBC) ▪ Hemosiderinuria
▪ Hemoglobinuria
• Site of hemolysis: ▪ Methemoglobinuria
o Intravascular • Release of Hgb directly in the plasma
▪ Happened inside the blood vessel Etiology
▪ Hgb directly released in plasma
• Mechanical injury
o Extravascular
o Thrombus formation in the blood vessel in cases of
▪ Normal; located commonly in the spleen disseminated intravascular coagulation
(graveyard of the senescent or old RBCs) o Defective prosthetic cardiac valves
 Normally, the body hemolyzes senescent ▪ RBCs will experience shear stress
RBCs so that the globin and iron portions will
be recycled for production of new RBCs that • Complement fixation (mismatch)
will circulate up to 120 days. o There is abnormal increase of sensitivity to
▪ Red cell destruction through phagocytosis of complement fixation, such as the absence of CD59
intact/fragmented red cells in the case of paraxosymal nocturnal
hemoglobinuria.

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 LECTURE UNIT 05: HEMOLYTIC ANEMIA (INTRINSIC DEFECTS)

o At night time, the blood pH becomes acidic which • Increase in indirect Bilirubin
triggers complement fixation and eventually lead to • Increase in urine urobilinogen & fecal urobilinogen
hemolysis of RBCs.
Table 1. Comparison of Intravascular and Extravascular
• Infection
Hemolysis
• Toxic injury
o Clostridial sepsis
Intravascular Extravascular
Laboratory Findings

• Serum Site of destruction

Within blood vessels Spleen or liver
of erythrocytes
o yellowish is the normal serum of
Activation of Cell-mediated
the patient (left)
Mechanism complement IgM or phagocytosis of IgM-
o the patient's serum indicates IgG or IgG-coated cells
hemolytic anemia (right)
Hemoglobinuria Positive direct
• Urine Laboratory Findings direct antiglobulin test antiglobulin test
o Hemoglobin from RBC is directly Hemosiderinuria Erythrocytes
released in the plasma → kidney
→ urine
o Patients with intravascular HEREDITARY ANEMIAS OF INCREASED RBC
hemolysis will eventually develop: DESTRUCTION
▪ hemoglobinuria • Mechanism: Hereditary RBC Membrane Abnormality
▪ hemoglobinemia.
o Heriditary spherocytosis
▪ Methemoglobinuria
▪ Albuminemia ▪ Increased number of spherocytes, RBCs, MCH,
▪ hemosiderinuria and MCHC
• High LD2 (LD1 Megaloblastic) – predominant o Heriditary elliptocytosis
isoenzyme in RBC
• Low Hemopexin, Haptoglobin ▪ RBCs are elliptical or oval-shaped

o carriers of heme and hemoglobin o Heriditary pyropoikilocytosis

o If there is increased hemolysis, it will exhaust the ▪ RBCs have abnormal sensitivity to temperature.
storage of these carriers ▪ E.g., Exposed to 45 C
Manifestation  normal RBCs will not lyse
 RBCs of patients with HP will typically lyse
• Jaundice between 45 - 46 C
o Yellowing of the skin o Heriditary stomatocytosis
o Hemoglobin → bilirubin
▪ There is channel defect, causing the appearance
▪ Increased levels of B1 or indirect bilirubin of mouth cells.
• Hemoglobinemia – directly released in the plasma o Heriditary xerocytosis
• Hemoglobinuria – filtered hemoglobin in the urine
• Hemosiderinuria
Hereditary Spherocytosis
o Increase in the release of hemoglobin and heme
leads to their precipitation and go to urine • Defect in RBC membrane protein composition
o Hemosidirin particles in the urine
o Spectrin
Extravascular Hemolysis ▪ Normally binds to actin
▪ Form dimers link at the end to end to form
• RBC destruction through phagocytosis of intact, catamers.
senescent/old RBC or fragmented red cells
• Most common site: spleen o Ankyrin – binds to spectrin fiber
o Band 3 protein - an integral protein
o Patients with increased extravascular hemolysis will o Protein 4.1 and 4.2 – mediates spectrin and ankyrin
manifest splenomegaly. linkages
• Anemia, jaundice • Presence numerous
• Will not present hemoglobinemia and hemoglobinuria microspherocytes in
Laboratory Findings peripheral blood smear.
• There are numerous
• High carbon monoxide (CO) – product of heme structural proteins in the
catabolism RBC membrane
o Carboxyhemoglobin

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 LECTURE UNIT 05: HEMOLYTIC ANEMIA (INTRINSIC DEFECTS)

• Spectrin dimers are linked together by another protein

called actin (grape-like structure)
o Composed of spectrin, alpha (α) and beta (β)
• Spectrin-actin linkages that connects spectrin and actin • When spherocytes go to splenic sinusoids, they are less
is by protein 4.1 and 4.2 deformable compared to biconcave shape
• Ankyrin binds the spectrin fiber and attaches integral
o Causes RBC trapping, and erythrostasis
membrane protein – band 3 (sausage-like structure)
o In spleen, it decreases in pH, and increase in
• Any abnormality in these structural proteins, eventually macrophage contact
cause the formation of spherocytes
▪ Spherocytes are big and once trapped, and
Defects exposed to decreased pH and increase in
macrophage contact → induce hemolysis
• Cytoskeletal defects
o If not hemolyzed, there can be further loss of
o SPTA1 & SPB (spectrin) membrane due to the presence of macrophages
o ANK (actin)
o EPB42 (Protein 4.2) ▪ It bites the RBC membrane, thereby lysing the
cell or there will be deformities producing bite
• Transmembrane protein defect cells
o SLC4A1 (band 3) • Microspherocytes have decrease survival in spleen
• Autosomal Dominant thereby causing hemolysis or defects

o Heterozygous o Defects, such as formation of bites once out of the

o Defective binding of spectrin to protein 4.1 spleen

▪ Spectrin and protein 4.1 interacts together Mechanism of Hereditary Spherocytosis

• Autosomal Recessive
o Homozygous spectrin deficiency
• Shows different
interactions of
cytoskeletal
transmembrane proteins
o Spectrin, band 4.1 &
4.2, band 3, and
ankyrin
o When there is a
defect in one of the
four proteins, there will be a formation of
spherocytes in hereditary spherocytosis
• When there is a defect in vertical membrane protein
interaction (spectrin, ankyrin, band 3, 4.1, & 4.2)
• (1) Skeletal membrane defect
o result to RBC losing its unsupported lipid membrane
• (2) Membrane instability
• Due to the local disconnection of lipid bilayer, and • (3) Membrane loss from Microvesicles loss
underlying cytoskeleton, this will cause the formation of • (4) Reduced surface-to-volume ratio
vesicles then the release of the microvesicles which
o From biconcave or discoid shape to spherocytes
results to:
o Causing spherocytosis
o losing some parts of cell membrane
o decrease in surface area to volume ratio of RBC, • (5) Poor cellular deformability
o changing the normal discoid or biconcave shape into • (6) Splenic trapping
a spherical one. • (7) RBC stasis, low glucose, low or acidic pH, and
increased macrophage contact
• (8) Hemolysis by phagocytosis or osmotic lysis,

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 LECTURE UNIT 05: HEMOLYTIC ANEMIA (INTRINSIC DEFECTS)

o ATP depletion due to trapping Summary

o acid-induced damage due to low pH
o macrophage processing due to its presence in
splenic sinusoids, oxidative damage
• (9) Produces another set of membrane loss

• The absence of “BAS” - Band 3.1, Ankyrin, and

Spectrin leads to the RBC and cytoskeletal defects
• There is an overactivity of sodium-potassium ATPase
o Microvesicle loss causing decrease in volume ratio
which might cause reduction in intracellular cations,
of RBC thereby producing spherocytes
causing more water to diffuse out of cell (dehydrated)
o Dehydration of cell causes increase in viscosity, and • Spherocytes
eventually leading to cellular dehydration o spleen → cannot squeeze in the splenic cord
(decrease deformability of spherocytes) → trapping
▪ deformability of spherocyte is decreased, since
of RBCs in splenic cords → increased contact to
they are prone to extravascular hemolysis by
acidic pH of spleen → increase contact to systemic
mechanism of phagocytosis or osmotic lysis
macrophages → induce hemolysis
 Osmotic injury is the dehydration of the cell
• Splenomegaly
Manifestation o Spleen will congest spherocytes due to their size
which makes them difficult to go out
• Anemia
o Px will undergone splenectomy
• Reticulocytosis
o Hemolysis is detected by BM to release more cells ▪ stops hemolysis but does not remove
spherocytes because the defect is not from the
• Marrow erythroid hyperplasia spleen but on the site of cytoskeletal proteins of
spherocytes.
o Due to increase in erythropoietin
▪ There will still be presence of spherocytes in the
• Mild jaundice blood smear since the defect is on the RBC and
• Spherocytes in PBS cytoskeletal membrane not on the spleen.
• Splenomegaly o Avoids over enlargement or splenomegaly
• Brown pigment stones
• Autosomal dominant - most common type
o When px developed anemia, associated with • Clinical Manifestations
splenomegaly, plus formation of bile or brown
pigment stones o Spherocytes on Peripheral Blood Smear
o (Brown) Gall stone / Bile stone
Peripheral Blood Smear o Jaundice

• Polychromatic ▪ Due to hemolysis = increased formation of

bilirubin
cells or
polychromasia o Splenomegaly
o Reticulocytes o Highly likely to be fatal in children
can appear as Laboratory Tests
polychromatic
RBC • OFT (Osmotic Fragility Test)
• Spherocytes or • EMA (Eosin-5-maleimide) binding test
small but full with • Autohemolysis test
Hgb spherocytes OFT (Osmotic Fragility Test)
o Once
spherocyte • Confirmatory test for hereditary spherocytosis
survives • Standard volume of fresh heparinized blood
splenic trapping, it causes the formation of bite cells
o Mix with NaCl solution ranging 0.85% isotonic saline
– 0.0 distilled water in 0.5-0.1 increments

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 LECTURE UNIT 05: HEMOLYTIC ANEMIA (INTRINSIC DEFECTS)

▪ Can be measured in 540nm wavelength • Uses fluorescent dye for Band 3, Rh, RhAg, and CD47
▪ Can also be seen thru naked eyes in the RBC membrane
• Spherocytes has limited capacity to expand in hypotonic • Uses flow cytometry – automated
solution and lysed at higher concentration of NaCl • Expected result: lower mean fluorescence intensity
(MFI)

o OFT result of normal RBC o MFI should be standardized

▪ Usually lyse between Autohemolysis Test

0.66%-0.38% • Spontaneous lysis of RBC at 37C for 48 hrs
• In vitro test
o Normal: RBCs acquire nutrients from plasma
o OFT result of spherocytic
o Autohemolysis test: ATP and glucose in the RBC
RBC (increased)
will be depleted → membrane loss → formation of
▪ has limited capacity to spherocytes
expand in hypotonic
▪ Stored blood bags are prone to hemolysis due to
solution due to their
running out of ATP and glucose and there will be
small volume ratio
formation of spherocytes
▪ lyse at higher NaCl concentration
▪ RBCs hemolysed at 0.85%
Table 2. Normal Values of Autohemolysis

Normal Values of Autohemolysis

With Glucose 0.2-2.0%

• Normal: Red cells + Sodium chloride Without Glucose 0-0.9%

o 0.72% - 0.52% = no hemolysis

o 0.48% (hypotonic solution) = start of hemolysis until
0.32% (displace complete hemolysis of RBC)

• Hereditary Spherocytes: Red cells + Sodium chloride

o 0.68% – RBCs start to hemolysed because there is
decrease capacity or ability to expand in hypotonic
solution
o RBCs of spherocytes easily bursts due to
cytoskeletal membrane defect and decrease surface • Without glucose, there will be hemolysis
volume ratio
o Hemolysis can be corrected with glucose or ATP
EMA (eosin-5-maleimide) binding test administration
• More sensitive as • Patient with Hereditary Spherocytosis
an alternative to
OFT o Without glucose, the hemolysis is complete
o With glucose or ATP – presence of hemolysis
• Suitable for low
volume pediatric ▪ If ATP or glucose is added, there will be
patient due to hemolysis (10-50%)
difficulty of ▪ There are also samples that will be hemolyzed
drawing them
blood o It can be corrected but not to the point of reference
interval when the glucose is added
• Can be performed within 3 hours
• Specimens are acceptable for analysis up to 7 days • Normal Patient
after collection
o without glucose - presence of hemolysis
o Delayed processing is possible o with glucose or ATP - Hemolysis is reduced
• Used to eliminate interference for transfuse or • The amount of hemolysis is noted after the incubation of
fragmented RBC the sample at 37°C for 48 hours

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 LECTURE UNIT 05: HEMOLYTIC ANEMIA (INTRINSIC DEFECTS)

Hereditary Elliptocytosis o Elliptocyte is somewhat derived from Spherocyte

o Flattened Spherocyte
• Associated with weakening of o Correspond to 10% of the cases of HE
the membrane skeleton o Manifest mild to moderate anemia
• Defective association of
proteins that hold the • Stomatocytic hereditary elliptocytosis
skeleton together o Rare
o Deficiency of Protein 4.1 o Only moderately oblong
o Create two central areas of pallor
▪ It could result to Spherocytic Hereditary o Erythrocytes
Elliptocytosis
▪ Both spherocyte and hereditary elliptocytosis ▪ Elongated
▪ Somewhat appears to be stomatocytic ovalocyte
o Spectrin dimer-dimer interaction
o Common in Southeast Asia - Southeast Asian
▪ Commonly involved in abnormal skeletal protein Ovalocytosis
interaction o Many cells will contain one or two transverse ridges
o Defective Ankyrin-Protein 3 interaction or a longitudinal slit (similar to stomatocytes)
o Associated with increased resistance to Malaria
▪ Causes the Atypical Hereditary Elliptocytosis
▪ Malarial parasites are sensitive
• Similar to Hereditary Spherocytosis ▪ They require perfect red blood cells to thrive
o From Hereditary Spherocytosis, it was pressed to ▪ Underlying defect is related to the deletion of the
27 bases of Band 3 gene
form Hereditary Elliptocytosis
• Defect: Abnormal skeletal protein interaction Laboratory Findings

o The abnormality is not the protein alone, but the • Normal OFT and autohemolysis
interaction between the proteins
o The association of spectrin and actin o Except for Spherocytic HE

▪ Involves the defect of the spectrin or the other ▪ Increased OFT

proteins in which it closely associates ▪ Positive for autohemolysis
• Increased B1 and fecal urobilinogen
Mechanism of Hemolysis • Depletion/decreased serum haptoglobin
• Same with Hereditary Spherocytosis • Peripheral blood smear -
elliptocytes
• Membrane Loss
• Decreased red cell deformability o Increased in oval or
• Increase rate of sodium efflux elongated red blood cell
• Causes shortened red blood cell survival shape
o Needs to be greater than
o Due to splenic trapping and destruction 25% RBC population for it
• Increased exposure to decrease pH (acidic) to be considered HE
• Increase contact time with macrophages • No treatment necessary - not
• Category of Anemia: Normocytic, Normochromic very severe
Anemia • For patients displaying moderate severe hemolytic
diseases
Clinical Variants of Hereditary Elliptocytosis
o Cases of spherocytic HE and stomatocytic HE
• Consists of 9 clinical variants
• Three major categories based on the grounds of clinical ▪ Can have splenectomy
severity and red cell morphology ▪ Elliptocyte will still persists because the problem
is cytoskeletal/ membrane defect
o Common hereditary elliptocytosis
o Spherocytic hereditary elliptocytosis Hereditary Pyropoikilocytosis
o Stomatocytic hereditary elliptocytosis
• “Pyropoikilocytes” -
• Common hereditary elliptocytosis increase sensitivity to
temperature
o Most prevalent
• Extremely rare
o Typical hereditary elliptocytosis
• Characterized by extreme
o Most are non-anemic (asymptomatic)
microcytosis, anisocytosis
o Minority have mild hemolysis
and poikilocytosis
o Least severe
• Defect: Abnormality in
• Spherocytic hereditary elliptocytosis spectrin dimer-dimer
association defect
o Significant number of rounded elliptocytes and
spherocyte o Similar in some forms of Hereditary Elliptocytosis
o Has a defect of Band 4.1

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 LECTURE UNIT 05: HEMOLYTIC ANEMIA (INTRINSIC DEFECTS)

o Formation of budding cells, fragmented cells,  increased contact with decreased in pH

spherocytes, elliptocytes and other bizarre shape  increased in splenic destruction and
cells. hemolysis.
• Autosomal recessive disorder • MCV: 150fL
• Represents subset of common hereditary elliptocytosis • Absence of a membrane protein located in the Band 7
• MCV: 25-55fL (Normal: 80-100 fL) region called stomatin.
• In peripheral blood smear, full of different types of cells
in various shapes and sizes Laboratory Findings

Mechanism of Hemolysis • Increased OFT and Autohemolysis test

• Rh Deficiency
• Membrane loss and rigidity o According to a study, individuals with Rh deficiency
o Similar to HS and HE syndrome might have the absence of Rh null or
markedly reduced of Rh mod usually has hemolytic
• Increased calcium inflow and outflow anemia with hereditary stomatocytosis.
o Increased influx of calcium o Patients with Rh deficiency:

• HPP red cells fragment at 45˚- 46˚ C ▪ Rh null or Rh mod asssociated with hereditary
stomatocytosis.
o Normal: RBC shows budding off and fragmentation
when heated to 49˚C Thromboembolic complication
o Patients with HPP
• Splenectomy
▪ red cells have membrane loss and rigidity which o It should be carefully
is less deformable. considered because
▪ There is thermal instability or when exposed to it is associated with
temperature such as 45˚- 46˚ C, they begin to increased risk of
fragment. thromboembolic
complication.
Laboratory Findings
▪ Formation of thrombus or clot
• Increased OFT and ▪ Thrombus or clot travels and the thrombus
Autohemolysis test becomes embolized.
• Image:
o Embolus - when the thrombus travels to different
o Not yet incubated at 45˚C sites.
(topmost)
o Results after incubation at ▪ When thrombus travels from the kidney going to
45˚C for 1 hour (lower) the brain.

▪ It becomes fragmented Hereditary Xerocytosis

and bud off similar to
yeast cells. • Autosomal dominant
disorder
o Similar to
Hereditary
Hereditary Stomatocytosis stomatocytosis
• Autosomal dominant • Most common form
disorder of stomatocytosis
• Characterized by
excessively permeable o Variant of stomatocyte
to Na+ and K+ at 37˚C • Result: Dehydration
• Result: Hydrocytosis
o Greater efflux of K+ than influx of Na+
o increased water
content inside the ▪ More potassium goes out from the cell than the
RBC sodium entering the cell
▪ It stimulates the water to go out from the cell
▪ When there is Na and K (solutes), water follows.
▪ Increased in cell volume without an increased in o Decreased cation cellular concentration leading to
membrane surface area which causes premature cellular dehydration
hemolysis. o Increased surface volume ratio
o Increased splenic destruction of swollen cells Laboratory Findings
▪ When they will be trapped in splenic sinusoid or • Stomatocytes
splenic chord and less deformable leads to:
o Less than 10%
 increase in splenic macrophage contact
o Hb: 8-10g/dL (Slightly lower than the normal

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 LECTURE UNIT 05: HEMOLYTIC ANEMIA (INTRINSIC DEFECTS)

• Target cells 𝑃𝐾
2 − 𝑝ℎ𝑜𝑠𝑝ℎ𝑜𝑒𝑛𝑜𝑙 𝑝𝑦𝑟𝑢𝑣𝑎𝑡𝑒 + 𝐴𝐷𝑃 → 𝑝𝑦𝑟𝑢𝑣𝑎𝑡𝑒 + 𝐴𝑇𝑃
o Predominance
o The hemoglobin concentrates o produced ATP is a form of energy
on one pole in the cell like ▪ No pyruvate kinase = no formation of ATP.
Mexican hat cell Therefore, increasing chance of RBC to die.
▪ There is target cells and • Glucose-6-phosphate
few poikilocytes
• In hereditary poikilocytosis, splenectomy can improve
the anemia because there will be no lyse of spherocyte.
If there is no spleen, there will be no extravascular
hemolysis.
o In hereditary xerocytosis, splenectomy does not
improve their anemia. Thus, it is contradicted
because it increases in thromboembolic
complication, similar to hereditary stomatocytosis
• Greater efflux of K+ than influx of Na+
o Stimulates H2O to go out
• Lab Findings:
o Stomatocytes
o Target cells
o NADPH is used to form reduced glutathione which is
• Osmotic fragility test
a potent antioxidant.
o Result: Abnormal especially after incubation
▪ If there are oxidant in the body in the form of
• Autohemolysis test reactive oxygen species (ROS), these can
trigger RBC hemolysis.
o Result: increased
▪ Without reduced glutathione, produced by the
o Hemolysis is not corrected with glucose. NADPH, patient is at risk of oxidative stress.
HEREDITARY ERYTHROCYTE ENZYMOPATHIES Red Blood Cell Metabolism
• Increase RBC destruction
o There is hereditary RBC enzyme deficiency
• Mechanism: Hereditary RBC enzyme deficiency

• Common RBC Enzymopathies

o G6PDD (Glucose-6-phosphate dehydrogenase
deficiency)
o PKD (Pyruvate kinase deficiency)
• These common RBC enzymes are important in the
maintenance of RBC membrane

• Hexose Monophosphate Pathway

o RBC will need glucose to live, glucose with the
• Pathway of pyruvate kinase in Glycolysis action of enzyme will be converted into Glucose-6-
Phosphate → 6-Phosphogluconate by the action of
o RBC need to maintain an adequate amount of ATP G-6-P Dehydrogenase to produce NADPH.
for it to function and not to die.

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 LECTURE UNIT 05: HEMOLYTIC ANEMIA (INTRINSIC DEFECTS)

▪ NADPH, is used by glutathione reductase to Laboratory Finding

produce glutathione from oxidized glutathione.
o Purpose of GSH (Reduced Glutathione). • Hemoglobin is decreased (3-4 g/dL) – Anemic
o Normocytic normochromic RBC type of Anemia
▪ Help glutathione peroxidase convert hydrogen
peroxide (a potent oxidant to prevent it from • Blood smear: Bite Cells and predomination of Heinz
destroying RBC) will be converted into 2 Bodies (only seen supravitally)
molecules of water and oxygen.
▪ Preventing oxidative stress to RBC o When heme is liberated from globin, and globin
denaturizes, this will form heinz bodies.
• Embden-Meyerhof Pathway o Oxidized hemoglobin from RBC → Methemoglobin,
o Fructose-6-Phosphate undergoes several ▪ sulfhydryl group and disulfide bridges will cause
conversions forming Pyruvic acid. decrease hemoglobin solubility
▪ Along the pathway pyruvate kinase is used.  precipitated in a form of heinz bodies,
o Without Pyruvate kinase, there will be no production o Heinz Bodies - Irreversible Membrane Damage
of 2 ATP
▪ When the denatured form of hemoglobin (Heinz
▪ No production of ATP = RBCs are prone to bodies) will adhere to the inner membrane of
Hemolysis RBCs
▪ RBC membrane is dependent to ATP (E.g., Na-K  Reaches to other areas of stress (i.e., blood
ATPase pump) for it to function, depletion will vessel) –intravascular hemolysis
cause influx of cations or anions in the RBC and  Reaches the spleen or the liver, it will
blood. accumulate causing macrophage interaction
Glucose-6-Phosphate Dehydrogenase Deficiency and phagocytosis – extravascular
hemolysis/pitting
• Most common enzymopathy w/ hemolysis
o Heinz bodies will go out of the blood vessel in the
• X- linked inherited diseases, common in women, form of bite cells which can be seen in the
homozygous, and inherited of anyone with large
peripheral blood
number of abnormal genes that code G6PD enzyme
• If with defect, or inherited abnormal genes →
susceptible to G6PD deficiency
o Decrease G6PD = oxidative denaturation of Hgb
▪ Denaturation of Hgb, is the major contributor to
the hemolytic process.

• Reticulocyte is increased (5x)

o Bite cells are inefficient in transporting oxygen thus
reticulocytes compensate in reduce oxygen
transport

Table 3. Classification of Glucose-6-Phosphate Dehydrogenase

Variants by the World Health Organization
• Glucose phosphate pathway uses G6PD enzyme → 6
phosphogluconate
G6PD
o This process will generate NADPH, need for Examples of
Class Enzyme Clinical Manifestations
production of reduced glutathione (GSH) → Variants
Activity
maintain RBC membrane
Severely
• If depletion of these enzyme, hemoglobin will be Chronic, hereditary
deficient:
oxidized to methemoglobin, iron in oxidized ferric state I <1% activity
nonspherocytic G6PD-Serres
is liberated from the globin which will be denatured hemolytic anemia; G6PD-Madrid
or not
forming Heinz bodies. severity is variable; rare
detectable
Severe, episodic acute
o RBC with Heinz bodies, will go to splenic sinusoid,
hemolytic anemia G6PD-
macrophages will undergo pitting resulting to bite Severely
associated with Mediterranean
cell figure of the RBC or golf ball/ pitted like II deficient:
infections and certain G6PD-
appearance. <10% activity
drugs, and fava beans; Chattam
not self-limited and may

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 LECTURE UNIT 05: HEMOLYTIC ANEMIA (INTRINSIC DEFECTS)

require transfusions o Attributed to the inefficient conjugation of the indirect

during hemolytic bilirubin by the liver (rather than the hemolysis)
episodes
• Chronic hereditary nonspherocytic hemolytic anemia
Mild to
moderately
Episodic acute hemolytic o Most patients are diagnosed at birth with neonatal
anemia associated with G6PD-A- hyperbilirubinemia which could continue in
III deficient:
infections and certain G6PD-Canton adulthood
10% to 60%
drugs; self-limited o No hemoglobinuria; some manifest acute episodes
activity
G6PD-B
Mildly
(wildtype) • Hemoglobinemia
deficient to • Hemoglobinuria
G6PD-A+
IV normal: 60% None
to 150%
(may also • There will be intravascular and extravascular hemolysis
manifest as
activity o Oxidation of globin chains
class III)
o Forming Heinz Bodies
Increased:
V >150% None Laboratory Diagnosis
activity
• Moderate to extremely
severe anemia
Manifestation o Normocytic,
normochromic anemia
• Typically, asymptomatic, unless exposed to oxidants o Depending on the
• Triggers of Hemolysis amount of oxidant taken
o Drugs or exposed
▪ Antimalarials (Chloroquine, Primaquine) • Marked anisocytosis and poikilocytosis
▪ Sulfonamides • Presence of: Bite cells and Heinz bodies
• Reticulocyte count = may reac h 30%
o Infections
• Pedigree of change in the
▪ Viral hepatitis, pneumonia, typhoid fever morphology varies on each
hemolytic episode
o Oxidants
o Heinz bodies cannot be
▪ fava beans (favism)
detected on Wright Stain
▪ moth balls, etc.
▪ Visualized using
• Common Manifestation:
Supravital stain (i.e.,
o Urine discoloration crystal violet, new methylene blue, brilliant cresyl
o Jaundice – icteric sclerae blue)
▪ Appears as dark purple inclusions attached to
• If presence of severe hemolysis, blood transfusion is the inner RBC membrane
needed
o Intravascular laboratory findings :
▪ Hemoglobinuria
▪ Hemoglobinemia
o Extravascular laboratory findings:
▪ Increased urobilinogen and stercobilinogen
▪ Increased bilirubin levels
Ascorbate Cyanide Test

• Incubation of blood with sodium

cyanide and sodium ascorbate
• Acute hemolytic anemia due to hemolysis secondary to (generation of hydrogen
drug exposure peroxide) → oxidation of Hb to
o Class 1-3 are clinically and hematologically normal methemoglobin = BROWN color
until the offending agent is taken 1-3 days after o Cyanide serves as a catalase
inhibitor
▪ Certain drugs have a threshold, but if taken in
excess it takes 1-3 days for hemoglobin to be ▪ Product is hydrogen
seen in the urine - hemoglobinuria peroxide
▪ RBC contains catalase
• Neonatal jaundice
which can interfere because it can detoxify
o “Neonatal hyperbilirubinemia” hydrogen peroxide
o 2-3 days after birth without concomitant anemia
o The brown color will develop after 1-2 hours after
incubation

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 LECTURE UNIT 05: HEMOLYTIC ANEMIA (INTRINSIC DEFECTS)

• Detection: Fluorescent screening • Most cases of G6PD deficient patients are

• Problem: It cannot differentiate the two most common asymptomatic and a self-limiting disorder
enzymopathies (G6PD and PK) o Due to continuous production of RBC in the bone
Fluorescent Spot Test marrow

• Generation of NADPH • Manifestations

– fluorescent light o Anemia
• Negative G6PD o Jaundice – increase bilirubin levels
deficiency o Hemoglobinuria – increase hemoglobin in urine
o If patient is G6PD o Hemoglobinemia – increase hemoglobin in blood
deficient, there will
Pyruvate Kinase Deficiency
be no or low
production of • Most common enzymopathy of the Embden-Meyerhof
NADPH Pathway (EMP)
▪ Cannot be detected (no fluorescence) • Autosomal recessive disorder with the mutation in the
Pyruvate Kinase LR (PKLR) gene
Quantitative Assay G6PD • Characterized by marked decrease in ATP production
• Spectrometry at 340 nm • Embden-Meyerhof Pathway
• Based on rate of reduction of NADP to NADPH o (1) Glucose will be converted to Glucose-6-
Phosphate
Autohemolysis Test
o (2) 90% of G6P will be converted into
• Slightly to moderately increased Glyceraldehyde-3-Phosphate
• Partially corrected by glucose o (3) Eventually, it will be converted into
• Same result with the hereditary elliptocytosis, and cases Phosphoenolpyruvate
of unstable hemoglobin o (4) By the action of the pyruvate kinase,
Phosphoenolpyruvate will be converted into
Summary of G6PD Deficiency Pyruvate
o (5) Two (2) ATPs are formed as a product of this
pathway.
• If there is a deficient level of PK, there would also be
decreased production of ATP.

• X-linked recessive disorder

• Most common in women
• Characterized by red blood cell hemolysis after
exposure to oxidative drugs, foods, and infection.
o Drugs: Aspirin, sulfonamides, nitrofurantoin, Results of PK Deficiency
dapsone, primaquine, quinidine
• K+ and H2O are lost from the cell
▪ Nitrofurantoin is used to treat urinary tract
infection o Loss of Potassium (K+) will cause an efflux wherein
it will also cause the release of water from the cell.
o Foods: Fava beans
o Infection • Cell shrinkage, distortion, and spiculation
• 2,3-DPG accumulates
• If patients are exposed with oxidizing agents, the red
cells will hemolyze and will form Heinz bodies. o If there is a deficiency of Pyruvate Kinase, there
would be no conversion of Phosphoenolpyruvate to
o Splenic macrophages will phagocytize the Pyruvate.
membrane of RBCs with Heinz bodies. Thereby o Thereby, causing now shunting of the
creating bite cells. glyceraldehyde-3P to become 2,3-DPG.

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 LECTURE UNIT 05: HEMOLYTIC ANEMIA (INTRINSIC DEFECTS)

o If there is an increase levels of 2,3-DPG, it will Quantitative Assay

cause Shift to the Right in the oxygen dissociation
curve. • Rate of NAD formation is proportional to PK activity
• Measured as a decrease in absorbance at 340 nm
▪ Low affinity of RBCs to oxygen • The enzyme pyruvate kinase catalyzes the following
▪ Results to release of oxygen reaction:
▪ patients with Pyruvate Kinase deficiency tends to
𝑃𝐾
tolerate the manifestations of anemia due to the 2 − 𝑝ℎ𝑜𝑠𝑝ℎ𝑜𝑒𝑛𝑜𝑙 𝑝𝑦𝑟𝑢𝑣𝑎𝑡𝑒 + 𝐴𝐷𝑃 → 𝑝𝑦𝑟𝑢𝑣𝑖𝑐 𝑎𝑐𝑖𝑑 + 𝐴𝑇𝑃
high levels of 2,3-DPG.
𝐿𝐷𝐻
Manifestations pyruvic acid + 𝑁𝐴𝐷𝐻 → 𝑙𝑎𝑐𝑡𝑖𝑐 𝑎𝑐𝑖𝑑 + 𝑁𝐴𝐷

• Anemia varies from

• NAD – Not fluorescent ↓absorbance at 340 nm
mild anemia to severe
neonatal anemia o More NAD produced = less fluorescence = more PK
• Jaundice activity
o Less NAD produced = increased fluorescence = less
o Hyperbilirubinemia PK activity
which requires
multiple • In quantitative PK assay:
transfusions to
o The hemolysate is prepared from the patient
fully compensated hemolytic process, especially to
anticoagulated blood after careful removal of WBC
adults.
▪ Removal of WBC is important because it has
▪ Adults are mild
high PK level → falsely increased PK result
▪ Children are more sensitive
▪ Contaminated WBC will have an increased PK
• Manifests chronic hemolysis – compensated, thus there result, and for patient with PK deficiency, this
will be adaptation kind of result will be interpreted as falsely normal
• Splenomegaly
• Result:
• Increased incidence of gallstones
o Normal: Fluorescence of NADH will disappear to the
o Due to excessive production of bilirubin formation of NAD, this will indicate normal PK
• Developed folate deficiency due to accelerated activity
erythropoiesis. o PKD: Increased fluorescence of NADH persists

o RBCs are prone to hemolysis

o Folic acid and Vitamin B12 are deficient in the HEREDITARY PLASMA CONSTITUENT
precursors ABNORMALITY
• Bone Marrow Aplasia • Abetalipoproteinemia
• Skin Ulcer • Lecithin-cholesterol acyltransferase (LCAT) deficiency
• During pregnancy, this carries the risk of fetal loss and
exacerbation of anemia in the mother. Abetalipoproteinemia

Laboratory Findings • Autosomal recessive malabsorptive disease

• Red cell survival is affected by the abnormality in the
• Normocytic, lipid metabolism
Normochromic • Mnemonics: 3A’s and 3R’s
• Reticulocyte count: 2.5 –
o 3A
15%
• Increased serum indirect ▪ Autosomal recessive
bilirubin ▪ Ataxia
▪ Acanthocytosis
o Due to intravascular
nature o 3R
• Decreased serum haptoglobin ▪ Retinis pigmentosa
• Increased urinary urobilinogen ▪ Retardation
▪ RBC shape
Autohemolysis Test alteration
• Increased • Lipid membrane of RBC is comprised of lipids
• Glucose: No effect
o If there is an abnormal absorption of lipids coming
• ATP: Corrects hemolysis
from the nutrients of food and plasma, there would
o In Hereditary Spherocytosis, the correction of be abetalipoproteinemia
hemolysis involves the addition of glucose and ATP
o In Pyruvate Kinase, the correction of hemolysis only Hereditary acanthocytosis
involves the addition of ATP.
• Autosomal recessive

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 LECTURE UNIT 05: HEMOLYTIC ANEMIA (INTRINSIC DEFECTS)

• Rare but serious disease • Decreased serum lipid levels

• Absence of b-lipoproteins in the serum apolipoprotein B o Plasma triglyceride levels are nearly 0
(apo B) o Very low from normal level
o Protein that coats chylomicrons
Lecithin-Cholesterol Acyltransferase (LCAT) Deficiency
• Mutation: MTP gene (Microsomal triglyceride transport
protein) • Rare inherited disorder
• LCAT – lipoprotein-associated enzyme which plays a
role in:
o Esterification of free cholesterol
o High-density lipoprotein maturation
o Intravascular stage of reverse cholesterol transport
pathway

Manifestation

• Nervous system and GI disorders

o Result to early death
• Malabsorption of fats, retinitis pigmentosa, neurologic
damage, RBC acanthocytosis • Absence of LCAT Decreased plasma cholesterol esters
o Neurologic damage – most problematic and increased free cholesterol in plasma

▪ Brain is comprised of lipids, and if MTP gene is Clinical findings

defective then it will lead to abetalipoproteinemia
• Renal disease
o Acanthocyte
o Increased free cholesterol in plasma thereby
▪ As a manifestation of deposited in renal tissues
profound disturbance
in the plasma
lipoprotein
▪ Irregularly
speculated RBC
resembling sea
urchin
Mechanism of hemolysis

• Membrane loss leading to membrane rigidity and less

deformability Normal Blackening (deposits)

o markedly decreased triglyceride and cholesterol in • Corneal opacities – common to Scandinavian families
the plasma
o Sphingomyelin level is increased-normal level in
plasma
▪ RBC membrane acquires increased
sphingomyelin = decreases fluidity, thereby
increasing membrane rigidity
• Mild anemia
 Rigid membrane → imbalance of the
sphingomyelin-lecithin ratio → decreased o Normocytic, normochromic type
RBC deformability, rigid → Increased rate of
cell destruction • Numerous target cells
• Hemolysis: Present
Laboratory findings
o Decreased erythropoiesis with the concurrent of
• Mild anemia renal disease
• Normal RBC indices o Decreased EPO

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