HEMATLOGY 2
Clinical Hematology
Midterm Topic 5- Anemia of Increased Destruction:
Intrinsic Defects
[BOOKS AND TRANS] TOPIC 5: ANEMIA OF INCREASED DESTRUCTION: INTRINSIC DEFECTS
OUTLINE
1) Introduction
2) Hereditary Red Blood Cell Membrane Abnormalities
3) Hereditary Spherocytosis
4) Hereditary Elliptocytosis
5) Hereditary Ovalocytosis (Southeast Asian Ovalocytosis)
A. Hereditary Pyropoikilocytosis
6) Overhydrated Hereditary Stomatocytosis
7) Dehydrated Hereditary Stomatocytosis
8) Other Hereditary Membrane Defects with Stomatocytes
A. Familial Pseudohyoerkalemia
B. Cryohydrocytosis
C. RH Deficiency Syndrome
9) Other Hereditary Membrane Defects with Acanthocytes
A. Neuroacanthocytosis
B. Abetalipoproteinemia (ABL)
C. Mcleod Syndrome
D. Chorea Acanthocytosis
10) Acquired Red Blood Cell Membrane Abnormalities
A. Stomatocytosis
B. Spur Cell Anemia
C. Paroxysmal Nocturnal Hemoglobinuria
11) RBC Enzymopathies
A. G6PD Deificiency
B. Pyruvate Kinase Deficiency
INTRODUCTION
 Intrinsic Hemolytic Anemia comprises of a large
group disorders which problem is in the Red Blood
Cells
 Intrinsic disorder can be divided into:
o Abnormalities of RBC Membrane
o Metabolic Enzymes or hemoglobin
 Most are HEREDITARY
o RED CELL MEMBRANE
 RBC MEMBRANE AND STRUCTURE AND
FUNCTION
 SHAPE: Biconcave discoid shape
o Two transmembrane protein complexes:
Vertical
 Ankyrin complex- link transmembrane
proteins, band 3 and RhAG (the
Rhassociated - glycoprotein) to the
cytoskeleton
 Protein complex- links transmembrane
proteins, glycophorin C, XK, Rh, and Duffy to
the cytoskeleton, and adducin and dematin
link with transmembrane proteins, band 3
and glucose transport (glut 1)
o Two major cytoskeletal proteins: a-spectrin and
b-spectrin antiparallel heterodimers:  Has worldwide distribution and affects 1 in 2000 to
HORIZONTAL
 The ability of spectrin repeats to unfold and
refold is likely to be one of the determinants
of membrane elasticity
 Cytoplasmic viscosity depends on the
concentration of haemoglobin
PADAYHAG, RIGIDOR S. | MLS-3D
RMT
2022
 A defect in the RBCs affects RBC
deformability and can result in premature
hemolysis and anemia
 LIFESPAN: 120 days
 Blood plasma serves as a barrier to separate the
vastly different ion and metabolite concentrations of
the interior of the RBC from its external environment
 Various membrane proteins also act as
o Receptors, RBC antigens, enzymes, and
support for the surface carbohydrates
(protective glycocalyx with the surface
glycolipids)
HEREDITARY RED BLOOD CELL MEMBRANE
ABNORMALITIES
 Hereditary are the most common defects in the RBC
membrane. However, acquired defects also exist
 Major disorders:
o Hereditary spherocytosis
o Hereditary elliptocytosis
o Hereditary pyropoikilocytosis
o Hereditary ovalocytosis
o Overhydrated hereditary stomatocytosis
o Dehydrated hereditary stomatocytosis
 Hereditary membrane defects can also be classified
as:
o Affect membrane structure (and alter geometry
and elasticity)
o Affect membrane transport (and alter
cytoplasmic viscosity
HEREDITARY SPHEROCYTOSIS
EPIDEMIOLOGY
 A heterogeneous group of hemolytic anemias
 It is characterized by numerous microspherocytic
erythrocytes on the blood film
3000 individuals of northern European ancestry
 In 75% of families, it is inherited as an autosomal
dominant trait and is expressed in heterozygotes who
have one affected parent.
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 HEMATLOGY 2
Clinical Hematology
Midterm Topic 5- Anemia of Increased Destruction:
Intrinsic Defects
 Homozygotes are rare; such patients present with
severe hemolytic anemia but have asymptomatic
parents
 In approximately 25% of cases, the inheritance is
nondominant, with some autosomal recessive cases
ETIOLOGY
 Genetic defect causes defects in membrane proteins
 Hemolysis results from o Interplay of intact spleen o
Intrinsic membrane protein defect
 Abnormal RBC morphology
PATHOPHYSIOLOGY
 HS erythrocytes are caused by membrane protein
defects resulting in cytoskeleton instability
 Spectrin deficiency leads to loss of erythrocyte
surface area, which produces spherical RBCs
 Spherocytic RBCs are culled rapidly from the
circulation by the spleen
 Splenomegaly Occurs
 There are 4 abnormalities of red cell membrane
proteins have been identified or the Mutated Gene
o 1. Ankyrin (ANK1)
o 2. Band 3 (SLC4A1)
o 3. Alpha-Spectrin (SPTA1)
o 4. Beta-Spectrin (SPTB)
o 5. Protein 4.2 (EPB42)
 Note: Each has a variety of mutations which
determines clinical presentation
 Two membrane protein defects that may cause HS:
o Defective binding of Spectrin to protein
4.1→autosomal dominant trait
 Spectrin Deficiency→ autosomal recessive
CLINICAL PRESENTATION AND PHYSICAL FINDING
 Three characteristics of clinical features of HS
 Anemia
o Usually mild-moderate
o Can be severe
 Jaundice
o Most common in newborns
o Exchange transfusion required at times
 Splenomegaly
o Detected in more than 75% of cases
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RMT
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CLINICAL LABORATORY FINDINGS AND
CORRELATION WITH THE DISEASE
COMPLETE BLOOD COUNT (CBC)
 Anemia is usually mild
 Adult Hb= >10g/dL
 Infants and young= <8-11g/dL
 MCV= between 77 and 87 fL (increased or markedly
increase)
 MCHC= increase inhald to two thirds of the patients
 Reticulocyte count= Between 5% and 20%
 RDW is generally normal; Aplastic crisis causes a
marked decrease in Hb and a low reticulocyte count
PERIPHERAL BLOOD FILM
 They appear as small and dark and lack of central
pallor
 Numerous microspherocytes are on the blood film o
Little poikilocytosis other than the spherocyte is seen
 The hallmark of HS is spherocytes on the peripheral
blood film.
BONE MARROW
 It displays erythroid hyperplasia
SPECIAL HEMATOLOGY TEST
 Osmotic Fragility test: Confirmatory test for HS
 Red cell in HS have increased Osmotic fragility
 Direct antiglobulin test is negative
 Autohemolysis test
CHEMISTRY
 Slight to moderate rise in unconjugated (indirect)
bilirubin, elevated fecal urobilinogen and decrease
haptoglobin levels may observed.
 The intravascular haemolytic indicators that does not
appear in Hs are Hemoglobinemia, hemoglobinuria
and hemosiderinuria
OSMOTIC FRAGILITY TESTING
 Principle: Osmotic Fragility reflects the shape of
erythrocytes. The osmotic fragility test demonstrates
increased RBC fragility in blood specimens in which
the RBCs have decreased surface area–to–volume
ratios.
 Specimen Requirement: Fresh heparinized blood
(15-20mm of defibrinated whole blood)
 Note: Anticoagulants containing oxalates are
undesirable because they alter pH
 Reagents: Saline concentrations; already
manufactured (e.g., the osmotic fragility kit produced
by Becton Dickinson, Rutherford, NJ)
 General Procedure:
o 1. Suspend red cells in a series of saline (NaCl)
solution ranging from 0.85%-0.1% and
incubate for 30 mins at room temp.
o 2. After each test suspension is centrifuged, the
absorbance of each supernatant is measured
at 540 nm.
o 3. Using this absorbance, percent hemolysis is
calculated by comparing each test solution to
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 HEMATLOGY 2
Clinical Hematology
Midterm Topic 5- Anemia of Increased Destruction:
Intrinsic Defects
the 0.85% NaCl solution wherein no hemolysis
is expected.
 Calculations: Calculation of hemolysis is based on the
absorbance of the supernant of each NaCl
concentration (Ax %)
 %Hemolysis=Ax% - A0.85% x 100%/ A0.0% -
A0.85%
INCUBATED OSMOTIC FRAGILITY
 Following 24-hour incubation
DIRECT ANTIGLOBULIN TEST
 The DAT detects antibody on the red cell surface, so
a negative result rules out immunemediated
hemolysis anemias and is expected in HS
EOSIN-5’-MALEIMIDE (EMA) BINDING TEST
 More sensitive alternative confirmation of HS
 It is a fluorescent dye that binds to transmembrane
proteins band 3, Rh, RhAg, and CD47 in the RBC
membrane
 Positive tests can also occur with congenital
dyserythropoietic anemia type II, Southeast Asian
ovalocytosis, and hereditary pyropoikilocytosis, but
these are rare condition
OTHER TESTS
 Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE)
COMPLICATIONS
 Patients may experience various crises, classified as
hemolytic, aplastic, and megaloblastic Patients with
moderate and severe HS can also develop folic acid
deficiency
 About half of patients, even those with mild disease,
also experience cholelithiasis (bilirubin stones in the
gallbladder or bile ducts) due to the chronic
hemolysis. Chronic ulceration or dermatitis of the legs
is a rare complication
EFFECTS OF TREATMENT ON LABORATORY
RESULTS
 Mild HS usually requires no treatment
 Splenectomy is reserved for moderate to severe
cases, and laparoscopy is the recommended method
 Major drawback is lifelong risk of overwhelming
sepsis and death from encapsulated bacteria and an
increased risk of cardiovascular disease with age
 Prior to splenectomy, patients receive vaccines for
pneumococcus, meningococcus, and H. influenza
type b, and postsplenectomy antibiotics may be
recommended
 Infants and young children are especially susceptible
to postsplenectomy sepsis, splenectomy is usually
postponed until after the age of 6 years
 After splenectomy, spherocytes are still apparent on
the blood film, and all of the typical changes in RBC
morphology seen after splenectomy also are
observed, including Howell-Jolly bodies, target cells,
and Pappenheimer bodies
PADAYHAG, RIGIDOR S. | MLS-3D
RMT
2022
DIFFERENTIAL DIAGNOSIS
 The immune disorders with spherocytes are usually
characterized by:
 POSITIVE RESULT: direct antiglobulin test (DAT)
 NEGATIVE RESULT: Hereditary Spherocytosis
 The eosin-5’-maleimide (EMA) binding test shows
decreased fluorescence typical of HS.
SUMMARY OF LAB FINDINGS
 Varies with severity of HS and ability of the bone
marrow to compensate for the hemolysis.
 As measured on some automated blood cell
analyzers.
o With some rare mutations, acanthocytes,
pincered cells, stomatocytes, or ovalocytes
may be seen in addition to spherocytes.
o A result within the reference interval does not
rule out HS; similar results can be observed in
conditions other than HS.
o Increased; decreased; SDS-PAGE, sodium
dodecyl sulfate-polyacrylamide gel
electrophoresis
 OTHER NOTES:
o Hereditary spherocytosis (HS) is caused by
mutations that disrupt the vertical membrane
protein interactions, which results in loss of
membrane, decrease in surface area–to–
volume ratio, and formation of spherocytes that
are destroyed in the spleen. Patients with HS
have anemia, splenomegaly, jaundice, an
increased MCHC, a negative DAT result,
biochemical evidence of hemolysis, and
spherocytes and polychromasia on the
peripheral blood film. RBCs in HS show
decreased fluorescence in the eosin-59-
maleimide binding test when measured by flow
cytometry, and the osmotic fragility test usually
shows increased fragility.
HEREDITARY ELLIPTOCYTOSIS
 HEREDITARY ELLIPTOCYTOSIS is a
heterogeneous group of disorder characterized by
large number of elliptical erythrocyte on the blood
film.
 Inheritance of hereditary elliptocytosis is autosomal
dominant.
ETIOLOGY AND PATHOPHYSIOLOGY
 HE results from gene mutation in which defective
proteins disrupt the horizontal in the protein
cytoskeleton and weaken the mechanical stability of
the membrane
 Mutations in atleast three genes
o SPTA1 which codes for alpha spectrin
o SPTB which codes for beta spectrin
o EPB41 which codes for protein 4.1
 The membrane structural defects involves spectrin or
proteins closely associates spectrin
o Three skeletal defects have been described
o deficiency of protein 4.1
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 HEMATLOGY 2
Clinical Hematology
Midterm Topic 5- Anemia of Increased Destruction:
Intrinsic Defects
o Spectrin dimer-dimer interaction
  the extent of disruption of the spectrin dimer
interaction seems to be associated to the
severity of clinical manifestation.
o Defective ankyrin-protein 3 interaction
 Neither the biochemical nor the structural lesions of
patients with HE correlates with the degree of
hemolysis.
 The mechanism of hemolysis involves membrane
loss, decreased red cell deformability and shortened
red cell survival due to splenic destruction.
 Heterozygous mutation= asymptomatic 
 Homozygous= moderate to severe hemolysis 
CLINICAL MANIFESTATION
 NO CLINICAL MANIFESTATION (MOST
AFFECTED PEOPLE ARE ASYMPTOMATIC)
 IF PRESENT; HEMOLYSIS(usually mild)
o ANEMIA
o JAUNDICE
o SPENOMEGALY
LABORATORY FINDINGS
 DISCOVERY OF HE IS OFTEN INCIDENTAL, THE
ASYMPTOMATIC PATIENT IS DIAGNOSED AFTER
PERIPHERAL BLOOD FILM IS EXAMINED FOR
ANOTHER CONDITION.
PERIPHERAL BLOOD SMEAR
 HE is elliptical or cigar-shaped (vary from few to
100%)
 Three types of HE have been described
o Common HE: most prevalent form
o Spherocytic HE: ≈10-20% cases
o Stomatocytic HE: rare
COMPLETE BLOOD COUNT
 HE are normochromic and normocytic
 Hb value is greater than 12 g/dl
 Reticulocyte count is less than 4%
BONE MARROW EXAMINATION
 Normoblast are normal in shape
 Erythyroid hyperplasia correlates with degree of
compensation for hemolysis
SPECIAL HEMATOLOGY TEST
 Osmotic fragility test: usually normal
 Autohemolysis test: usually normal (in cases of
spherocytic HE, values of these test are increased)
CHEMISTRY
 Increased unconjugated bilirubin and fecal
urobilinogen
 Decreased serum haptoglobin( be observed when
hemolysis is present).
TREATMENT
 ASYMPTOMATIC- REQUIRES NO TREATMENT
 SIGNIFICANTLY ANEMIC AND SHOWS SIGN OF
HEMOLYSIS- SPLENECTOMY
 PATIENTS WITH LIFE THREATENING ANEMIA-
TRANSFUSION

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RMT
2022
HEREDITARY PYROPOIKILOCYTOSIS
 An autosomal recessive disorder of the red blood cell
(RBC) membrane that is clinically related to, and is
considered a subtype of hereditary elliptocytosis
(HE).
 Involves a functional defect in spectrin, which is the
major cytoskeletal protein of the RBC cell membrane
thus calling it as a red cell structural abnormality. - It
manifests as a severe hemolytic anemia with thermal
instability of the red blood cells.
 ETIOLOGY AND PATHOPHYSIOLOGY
 Hereditary pyropoikilocytosis (HPP) is subtype of
hereditary elliptocytosis (HE), a red blood cell (RBC)
membrane disorder that results from mutations in the
genes
 Encoding α-spectrin (SPTA1), β-spectrin (SPTB), or
protein 4.1R (EPB41) which leads to both extreme
morphologic abnormalities and thermal instability.
 HPP has an autosomal recessive inheritance and is
typically caused by biallelic (homozygous or
compound heterozygous) mutations.
 HPP is a disease emanating from a defect in spectrin,
which is the major peripheral protein of the red blood
cell (RBC) membrane.
 HPP is characterized by an abnormality in both
horizontal (spectrin self-association defect) and
vertical (spectrin deficiency) RBC membrane protein
interaction.
 This blood disorder is characterized by an RBC
morphology similar to that seen in patients suffering
from extensive burns— hence the term
pyropoikilocytes.
 This condition may result in severe hemolytic anemia
as the RBC's lipid membrane destabilizes and loses
its ability to withstand intravascular shear stress.
 HPP red cells also are characterized by elevated
calcium content with increased inflow and reduced
outflow of calcium, but this abnormality appears to be
secondary to the membrane structural defect.
CLINICAL PRESENTATION
 HPP is considered the most severe type of hereditary
elliptocytosis (HE) and manifests during infancy.
 It is generally recognized when an infant or young
child presents with a transfusion-dependent
hemolytic anemia.
 The family history typically includes nonhemolytic
hereditary elliptocytosis in a parent or sibling.
 The other parent likely has a previously undiagnosed
spectrin deficiency.
 Most patients are of African origin, though cases have
been reported in people of Arabian or Caucasian
descent.
 Physical examination is remarkable for signs of
anemia.
 Signs of extra medullary haematopoiesis such as
frontal bossing and splenomegaly could be
appreciated.
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 HEMATLOGY 2
Clinical Hematology
Midterm Topic 5- Anemia of Increased Destruction:
Intrinsic Defects
 Affected children can have growth retardation.
 The chronic hemolytic disease can be complicated by
gallbladder disease.
LABORATORY FINDINGS AND CORRELATIONS
 A complete blood count and a peripheral blood smear
are the most important studies to obtain when testing
for HPP.
 The peripheral blood smear of a patient with HPP
demonstrates extreme poikilocytosis with
fragmentation, microspherocytosis, and elliptocytosis
similar to that in patients with thermal burns.
 The mean cell volume (MCV) is very low (50 to 65 fL
fr. Rodak, 25-55 fL fr. Steiniger) because of the RBC
fragments.
 RBCs in the HPP phenotype show marked thermal
sensitivity.
 CBC: Extreme microcytosis
 Hemoglobin is decreased in proportion to the degree
of hemolysis
 Reticulocyte counts are elevated
 RPI > 3, normocytic/normochromic indices
 Results in hereditary pyropoikilocytosis include the
following:
o Increased osmotic fragility
o Decreased acid glycerol lysis time
o Marked decrease in the maximum value of the
deformability index (DI max), with a distorted
trapezoidal profile, on osmotic gradient
ektacytometry
 Markedly decreased fluorescence on the eosin-5′-
maleimide (EMA) binding test
 Markedly decreased mean corpuscular volume -
microspherocytosis
 Thermal sensitivity testing shows fragmentation of the
red blood cells at temperatures as low as 45°C.
SPECIAL TESTS
 Osmotic gradient ektacytometry has been the
reference technique for diagnosis of RBC membrane
disorders, but its limited availability has severely
restricted its use.
 An ektacytometer is a laser-diffraction viscometer in
which deformability is measured as a continuous
function of the osmolality of the suspending medium
 Evaluation of the ratio of spectrin to band 3 to
determine the spectrin level so that this entity can be
differentiated from homozygous hemolytic
elliptocytosis.
 Ultrasound of the biliary tract may show gallstones,
especially in patients with symptomatic cholelithiasis.
If ultrasonography is nondiagnostic, a nuclear scan
can be done to work up gallbladder disease.
 Thermal Sensitivity Testing shows fragmentation of
the red blood cells at temperatures as low as 45°C.
DIFFERENTIAL DIAGNOSIS
 The diagnosis of HPP relies on identifying abnormal
red blood cell (RBC) morphology on peripheral blood
smear and identifying characteristic membrane
PADAYHAG, RIGIDOR S. | MLS-3D
RMT
2022
biomechanical properties using osmotic gradient
ektacytometry.
 The diagnosis can often be made by reviewing the
patient's clinical/family history, blood count results,
reticulocyte count, red cell morphology, and
chemistry results, together with tests available
through hematology laboratories.
 In neonatal poikilocytosis, which occurs almost
exclusively in African-American families, newborns
and infants have severe hemolytic anemia that
typically resolves after the first year of life.
Transfusions and phototherapy may be required
during severe hemolytic anemia and jaundice. The
resolution of symptoms after a year helps distinguish
neonatal poikilocytosis from hereditary
pyropoikilocytosis.
 To distinguish it from HE, the blood smear must have
elliptocytes, as seen in hereditary elliptocytosis,
together with the following:
o Microcytic red cells
o Polychromasia
o Bizarrely shaped cells with fragmentation,
poikilocytes, pyknocytes, and
microspherocytes
 To differentiate it from Hereditary Spherocytosis, the
red blood cells must be distinguished from that of
round, densely staining red cells that lack central
pallor and have a smaller than normal diameter and
the absence of frontal bossing as clinical
manifestation
 To differentiate it from Southeast Asian ovalocytosis,
the blood smear must have extreme poikilocytosis
with fragmentation, microspherocytosis, and
elliptocytosis or cegar/pencil shaped RBCs instead of
oval/egg-shaped erythrocytes.
DIFFERENTIAL DIAGNOSES
 Hereditary Elliptocytosis
 Hereditary Spherocytosis
 Neonatal poikilocytosis
 Southeast asian ovalocytosis
 All of these conditions manifest polychromasia, RPI is
greater than 3 and has normocytic/normochromic
indices thus these parameters cannot be used to
differentiate such.
TREATMENT AND MANAGEMENT
 No specific medications are used to treat patients with
hereditary pyropoikilocytosis. The need for treatment
with medications is patient-specific and based on
individual complications.
 In the setting of severe hemolytic anemia with
hereditary pyropoikilocytosis, transfusion of packed
red blood cells is necessary.
 Folate supplementation is also necessary in chronic
hemolytic disease. Folic acid is often used to prevent
folic acid deficiency, which may occur as a result of
increased erythropoiesis.
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 HEMATLOGY 2
Clinical Hematology
Midterm Topic 5- Anemia of Increased Destruction:
Intrinsic Defects
 In the acute care setting, supportive measures (eg,
intravenous fluids, oxygen, monitoring of blood
counts) are provided according to the needs of the
individual patient.
 Iron chelation therapy may be necessary in patients
who develop significant iron overload from red blood
cell transfusions.
 The spleen is the primary site of red cell sequestration
and contribute to the severity of anemia in patients
with HPP.
 Splenectomy has been advocated by some experts
for severe hemolytic anemia refractory to or
complicated by chronic blood trasnfusion.
SOUTHEAST ASIAN OVALOCYTOSIS
 Southeast Asian ovalocytosis (SAO) is a rare
hereditary red cell membrane defect characterized by
the presence of oval-shaped erythrocytes and with
most patients being asymptomatic or occasionally
manifesting with mild symptoms such as pallor,
jaundice, anemia and gallstones.
 It is a form of hereditary elliptocytosis
 Condition caused by a mutation in the gene for band
3 that results in increased rigidity of the membrane
and resistance to invasion by malaria because of
alterations in band 3, which is one of the malaria
receptors.
 It is common in the malaria belt of Southeast Asia,
where its prevalence can reach 30%.
 The inheritance pattern is autosomal dominant, and
all patients identified are heterozygous.
ETIOLOGY AND PATHOPHYSIOLOGY
 SAO results from a 27 bp deletion in the SLC4A1
gene, localized on chromosome 17q21.31
(SLC4A1del27 mutation).
 Deletion occurs at the interface between the
transmembrane and cytoplasmic domains.
 This gene codes for a band 3 anion transport protein
which is the bicarbonate/chloride exchanger in red
blood cell membranes and defects in this protein
cause membrane rigidity.
 The mutation causes an increase in membrane
rigidity that may be due to tighter binding of band 3 to
ankyrin or decreased lateral mobility of band 3 in the
membrane.
 RBC membranes also have decreased elasticity as
measured by ektacytometry and micropipette
aspiration.
 The association of mutation SLC4A1del27 with other
mutations in the SLC4A1 gene may result in distal
renal tubular acidosis associated with SAO in some
cases.
CLINICAL PRESENTATION
 Newborns with SAO might be symptomatic with
hemolysis at birth that leads to anemia, pallor or
jaundice.
PADAYHAG, RIGIDOR S. | MLS-3D
RMT
2022
 Anemia and hyperbilirubinemia in neonates is
common together with mild splenomegaly and
gallstones.
 Hemolysis usually disappears in the first three years
of life.
 Adults are asymptomatic or have only minimal
hemolytic anemia.
 Resistance to malaria is also noted.
 Prevalent in some areas of Southeast Asia
LABORATORY FINDINGS AND CORRELATIONS
 In patients with SAO, hemolysis is mild or absent.
 On a peripheral blood film, typical cells of SAO are
oval RBCs with one to two transverse bars or ridges
and usually comprise about 30% of the RBCs.
 Some of them are stomatocytic with more than one
stoma, in the absence of hemolysis.
SPECIAL TESTS
 Genetic assays can also be used to identify the
mutation in the SLC4A1 gene.
 However, the molecular mechanism responsible for
the increased membrane rigidity that results from the
band 3 mutation has not yet been elucidated up to this
day.
DIFFERENTIAL DIAGNOSIS
 Differential diagnosis includes hereditary
elliptocytosis which can be differentiated by having
oval RBCs with one to two transverse bars or ridges
and usually comprise about 30% of the RBCs in the
peripheral blood film of SAO
 Hereditary spherocytosis as this condition shows
round, densely staining red cells that lack central
pallor instead of an oval-shaped RBC
 Hereditary stomatocytosis as this disorder is
characterized by red blood cell (RBC) dehydration
and lysis due to leaking sodium and potassium ions.
 In SAO, it is the bicarbonate/chloride exchanger
defect that causes membrane rigidity
 Same with HPP, all of these conditions manifest
polychromasia, RPI is greater than 3 and has
normocytic/normochromic indices thus these
parameters cannot be used to differentiate such.
TREATMENT AND MANAGEMENT
 Most cases of SAO are asymptomatic and treatment
is not necessary.
OVERHYDRATED HEREDITARY STOMATOCYTOSIS
 Other name: Hydrocytosis
 Membrane cation permeability defect
 Overhydrated RBCs
 Autosomal dominant
ETIOLOGY AND PATHOPHYSIOLOGY
 RHAG gene mutation
o Codes for Rhag protein
 Rhag protein deficiency
o Transmembrane protein
o Ankyrin complex component
 Secondary deficiency of stomatin ( transmembrane
protein)
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 HEMATLOGY 2
Clinical Hematology
RMT
Midterm Topic 5- Anemia of Increased Destruction:
Intrinsic Defects
2022
o Participate in ion channel regulation NOTE
 Swollen cells  Hereditary Stomatocytosis
o Excessive permeability to sodium & potassium o Increased MCV
@ 37˚C o Decrease d MCHC
o Increased influx of sodium into the cells  Hereditary Xerocytes
Potassium loss is exceeded o Increased MCV
o Decreased density & viscosity of cytoplasm o Decrease d MCHC
o Increased cell volume OTHER HEREDITARY MEMBRANE DEFECTS WITH
o No membrane deformability STOMATOCYTES
CLINICAL PRESENTATION FAMILIAL PSEUDOHYPERKALEMIA
 Moderate to severe hemolytic anemia  Leaky red blood cell
LABORATORY FINDINGS & CORRELATIONS  First described by Stewart
 Stomatocytes  An inherited, mild, non-haemolytic subtype of
 Macrocytes hereditary stomatocytosis
 Decreased MCHC  Stored below 37°C.
 Reticulocytosis  Reported as an Autosomal dominant inherited
 Elevated RBC sodium concentration disorder
 Increased net cation concentration CLINICAL DESCRIPTION
 Increased Osmotic fragility  Asymptomatic condition
TREATMENT & MANAGEMENT CAUSE:
 Splenectomy
DEHYDRATED HEREDITARY STOMATOCYTOSIS  Increased passive potassium leakage at low
 Other name: Hereditary Xerocytosis temperatures
 Defect in membrane cation permeability TREATMENT:
 Dehydrated RBCs  No treatment required
 Most common form of stomatocytosis LABORATORY FINDINGS:
 Autosomal dominant  CBC parameters are near the reference intervals
ETIOLOGY & PATHOPHYSIOLOGY Occasional stomatocytes may be observed on the
 PIEZO1 gene mutation peripheral blood film.
o Codes for Piezo-type mechanosensitive ion TREATMENT/MANAGEMENT:
channel component 1 protein in the RBC  No treatment is required. Correct diagnosis is
membrane important to avoid misplaced insulin/glucose and/or
 PIEZO1 protein combines with other proteins to form fludrocortisone treatment for supposed true
a pore in a channel to mediate cation transport o hyperkalemia.
Membrane is excessively permeable to potassium o CRYOHYDROCYTOSIS
Potassium leaks out of the cell  Also known as hereditary cryohydrocytosis with
 Increased phosphatidylcholine normal stomatin
 Diminished spectrin phosphorylation  Rare disorder that manifests as a mild to moderate
o Target cells hemolytic anemia with leakage of sodium and
o Hemoglobin conc at one pole of the cell potassium from the RBCs and stomatocytosis
o No membrane deformability  The RBCs have marked cell swelling and hemolysis
CLINICAL PRESENTATION when stored at 4° C for 24 to 48 hours.
 Mild to moderate anemia  A subtype of hereditary stomatocytosis
 Jaundice  Patients suffer from mild to moderate hemolysis, with
 Mild to moderate splenomegaly or without anemia
 Fetal loss  SLC4A1 (Solute Carrier Family 4 Member 1 (Diego
 Hydrops fetalis Blood Group)
 Neonatal hepatits  Transport of glucose and other sugars, bile salts and
LABORATORY FINDINGS organic acids, metal ions and amine compounds.
 Reticulocytosis TREATMENT AND MANAGEMENT
 Elevated MCHC
 Treatment of the RBC of a patient with CHC with high
 Decreased osmotic fragility
doses of 4-acetamido-4′- isothiocyanato-2,2′-
 Decreased net cation concentration in the RBCs
stilbenedisulfonic acid disodium salt hydrate (SITS) to
TREATMENT & MANAGEMENT
decreased the membrane permeability to K+
 Do not require treatment
RH DEFICIENCY SYNDROME
 Not improved by splenectomy
 Also known as rh-null syndrome

PADAYHAG, RIGIDOR S. | MLS-3D 7

 HEMATLOGY 2
Clinical Hematology
Midterm Topic 5- Anemia of Increased Destruction:
Intrinsic Defects
 Comprises a group of rare hereditary conditions in
which the expression of Rh membrane proteins is
absent (Rh-null) or decreased (Rh-mod).
 Two types of Rh deficiency syndrome:
o The regulator types
o The amorph type
ETIOLOGY:
 Genetics: Inherited in an autosomal recessive
manner
CLINICAL DESCRIPTION:
 Mild to moderate hemolytic anemia
 Stomatocytes and occasional spherocytes may be
observed on the peripheral blood film
TREATMENT
 Symptomatic patients may be treated splenectomy
OTHER HEREDITARY MEMBRANE DEFECTS WITH
ACANTHOCYTES
NEUROACANTHOCYTOSIS
 Term used to describe a group of rare inherited
disorders characterized by neurologic impairment
and acanthocytes on the peripheral blood film.
 Neuroacanthocytosis syndromes are rare
 Underdiagnosed due to their low prevalence and high
clinical variability
MORPHOLOGY:
 Starlike appearance with spiky or thorny appearing
projections
 Three disorders characterized
neuroacanthocytosis:
ABETALIPOPROTEINEMIA (ABL):
 Rare autosomal recessive disorder characterized by
fat malabsorption, progressive ataxia, neuropathy,
retinitis pigmentosa, and acanthocytosis.
 ETIOLOGY AND PATHOPHYSIOLOGY
o Caused by mutations in the MTP (microsomal
triglyceride transfer protein) gene. By which,
MTP gene is needed to transfer and assemble
lipids onto apolipoprotein
 CLINICAL PRESENTATION AND LABORATORY
FINDINGS
o Acanthocyte is the hallmark feature of this
disease.
o Triglyceride and cholesterol are decreased;
sphingomyelin is increased in the plasma.
o Affected individuals have mild hemolytic
anemia, normal RBC indices and normal to
slightly elevated reticulocyte count.
o Patients with ABL manifest: Steatorrhea and
failure to thrive in young children (due to fat
malabsorption) or; Ataxia and neuropathy in
young adults (due to decreased absorption of
fat-soluble vitamin E).
 TREATMENT
o High doses of Vitamin A and E (reduce the
neuropathy and retinopathy)
PADAYHAG, RIGIDOR S. | MLS-3D
RMT
2022
MCLEOD SYNDROME (MLS)
 A neurological disorder that occurs almost exclusively
in boys and men.
 This disorder affects movement in many parts of the
body.
 ETIOLOGY AND PATHOPHYSIOLOGY
o An X-linked disorder caused by mutations in KX
gene.
o Men who lacks Kx on their RBCs have reduced
expression of Kell antigens, reduced RBC
deformability and shortened RBC survival.
o The Kx protein is found in various tissues,
particularly the brain, muscle, and heart.
o KX codes for the Kx protein, a membrane
precursor of the Kell blood group antigens.
o The absence of Kx antigen and reduction of
Kell antigen is known as the McLeod
phenotype.
 CLINICAL PRESENTATION AND LABORATORY
FINDINGS
o Patients with MLS have variable
acanthocytosis up to 85%, mild anemia, and
late-onset (aged 40 to 60 years), slowly
progressive chorea, peripheral neuropathy,
myopathy and neuropsychiatric manifestations.
o Some female heterozygote carriers may have
acanthocytes but neurologic symptoms are
rare and clinical manifestations depend on the
by proportion of RBCs with the normal X
chromosome inactivated versus those with the
mutant X chromosome inactivated.
 TREATMENT AND MANAGEMENT
o No current treatments available to prevent or
slow the progression of McLeod
neuroacanthocytosis syndrome.
o Antipsychotics:
 Decrease the involuntary movements.
o Botulinum toxin injections:
 Improve symptoms of dystonia.
Anticonvulsants and antidepressants: o May
treat seizures
CHOREA ACANTHOCYTOSIS:
 Rare autosomal recessive disorder characterized by
chorea, hyperkinesia, cognitive impairments and
neuropsychiatric symptoms.
 ETIOLOGY AND PATHOPHYSIOLOGY
 Caused by the mutation in VPS13A, a gene that
codes for chorein.
 Chorein is a protein which may be involved in
trafficking proteins to the cell membrane.
 A deficiency of chorein may lead to abnormal
membrane protein structure and acanthocyte. The
underlying pathophysiology of ChAc is not yet fully
deciphered.
8
 HEMATLOGY 2
Clinical Hematology
RMT
Midterm Topic 5- Anemia of Increased Destruction:
Intrinsic Defects
2022
 CLINICAL PRESENTATION AND LABORATORY  Markedly Increase Cell Sodium
FINDINGS  Reduce serum haptoglobin concentration (for
o The mean age of onset of the neurologic children or aldult).
symptoms is 35 years. In patients with ChAc, TREATMENT AND MANAGEMENT
5%-50% of RBCs on the peripheral blood film  No SPECIFIC TREATMENT but can be manage by
are acanthocytes. regulating the alcohol intake.
 TREATMENT AND MANAGEMENT SPURR CELL ANEMIA
o No current treatments
 “Spurr Cells”
o Management includes:
 These are small densely stained red cells that are no
 Botulinum toxin for decreasing the dystonia
longer disc shaped and have few irregularly spaced,
Phenytoin, clobazam, and valproate for
pointed spicules or thornlike projections.
seizure management; and antidepressant or
 Defect in lipid or protein composition of the RBCs
antipsychotic medications.
ACQUIRED RED BLOOD CELL MEMBRANE  Spheroid with 3-12 irregular spikes
ABNORMALITIES  Appearance: Clublike
 RBC membrane is freely permeable to water & anion; ETIOLOGY AND PATHOPHYSIOLOGY
Chloride & Bicarbonate can traverse the membrane  Defect/changes: cause by changes in the ratio of
in less than a second plasma lipids (lecithin and sphingomyelins)
 RBC membrane is impermeable to sodium &  In which the free cholesterol accumulates in the outer
potassium leaflet of the RBC.
STOMATOCYTOSIS  Spleen remodels the membrane of the RBCs into
 Also known as “Coffee-bean or Kissing Lips” long, rigid projection because of the remodeling,
 Stoma means: Mouth, Slit, Pore becomes entrapped and hemolyzed in the spleen,
 Shape: Bowl Shape o occur frequently as “Drying CLINICAL PRESENTATIONS
artifacts”  Hemolytic Anemia
 These are type of erythrocyte that has a central slit or  Anorexia
stoma instead of circular area of pallor.  Jaundice
 3-5% of normal individuals may have stomatocyte.  Splenomegaly
And they contribute hemolytic anemia LABORATORY FINDINGS AND CORRELATIONS
ETIOLOGY AND PATHOPHYSIOLOGY  Abetalipoproteinemia
 These are due to the alteration in the permeability  Severe liver disease.
(Osmotic changes) of cells membrane which SPECIAL TEST
increases the cell volume,  Microscopic Examination
 Manipulative flow of Sodium and Potassium inside TREATMENT AND MANAGEMENT
the cells. – Passive leak. (Hypotonic)  Liver transplant
 Primarily the unregulated/uncontrolled Na/K+ Jump. PAROXYSMAL NOCTURNAL HEMOGLOBINURIA
CLINICAL PRESENTATIONS
 It is a rare type of chronic intravascular hemolytic
 Acute Hemolytic Anemia o Fatigue anemia caused by an “Acquired clonal hematopoietic
LABORATORY FINDINGS AND CORRELATIONS stem cell mutation that results in circulating RBCs that
 Alcohol liver diseases lacks Glycosylphosphatidylinositol (GPI). (
 Artifact – cup shape Glycosylphosphatidylinositol Structure (Rodaks))
SPECIAL TESTS  Compose of –
 Microscopic Examination o Phosphatidylin ositol
o Glycan Core
 Reticulocyte count test
o Glucosamine
DIFFERENTIAL DIAGNOSIS
o 3 Mannos Residues
 Acquired: hemolytic anemia occurs primarily with o Ethanolamine Phosphate
recent excessive alcohol ingestion. The PATHOPHYSIOLOGY
approximately the visible stomatocyte in microscopic
 X-Chromosome codes for PIGA Gene that codes for
examination is less than 5%.
o Normal or Slight Increase of Cell Sodium Phosphatidylinositol N-
acetylglucosaminyltransferase subunit-A (PIGA-A)
 Heredity: if the percentage of stomatocyte in
 Dysfunction of the glycosyl transferase enzyme
microscopic examination range from 30- 50% in
results in deficiency of the GPI Anchors.
patient with heredity stomatocytes.
o Elevated reticulocyte count  GPI Anchors are complement-regulators and these
o Elevated serum bilirubin are CD55 (Decay-accelerating factor)) &
CD59(membrane inhibitor of reactive lysis).
PADAYHAG, RIGIDOR S. | MLS-3D 9
 HEMATLOGY 2
Clinical Hematology
RMT
Midterm Topic 5- Anemia of Increased Destruction:
Intrinsic Defects
2022
 CD55 – inhibits the activation of the complement INTERPRETATION
alternative pathway C3 and C5 convertases.  Multiparameter Flow Cytometer (New Confirmatory
 CD59 – Prevents the formation of Membrane attack test). Altus. CO by -Mam Bandoy
complex (MAC).  Principle: Detects the absence of the GPI-Anchored
CLINICAL PRESENTATION (STEININGER AND proteins on WBCs instead of RBCs
RODAKS)  Greater accuracy than using flow cytometry method.
 Hemolysis but asymptomatic  Uses: Fluorescent monoclonal antibodies GPI-
 Thrombosis and Bone Marrow Failure (cause by anchored proteins such as:
acquired aplastic anemia or myelodysplastic o (CD59, CD55, CD24, CD16, CD66b, CD14). –
syndrome) IMPORTANT!!
 Esophageal spasms and Dysphagia o Along with non-GPI anchored proteins such as:
 Slight jaundice (RBC breakdown), abdominal pain  (CD15, CD33, CD64)
 Splenomegaly o To Identify granulocytes and monocytes.
 Unusual Hepatomegaly o REMEMBER!! Granulocyte: CD24, CD15,
CD45
 Hemoglobinuria - @ Night, when pH falls down
o Monocyte: CD14, CD64, CD45
LABORATORY FINDINGS AND CORRELATION
 WAS ABLE TO DETECT GPI-DEFICIENT!! WITH
 Decrease hemoglobin levels (may drop <6g/dL) SENSITIVTITY OF 0.01%
 RBCs may in vary from macrocytic, normochromic to  Flourescein-labaled proaerolysin variant (FLAER)
microcytic, TYPE I
 Hypochromic (Increase iron loss)  Normal RBCs, normal expression of CD55 and CD59
 Thrombocytopenia (Lifespan of the platelet is not and, little or no complement mediated hemolysis
affected)  Normal expression of CD59 will show High Intensity
SPECIAL TESTS of fluorescence.
 Sugar water or Sucrose lysis screening test TYPE II
(Screening test)  Results to the PIG-A mutation that cause only a
 Principle: Strains the PNH erythrocyte if they will partial deficiency CD55 and 59 and are relatively
remain intact or not by adding low ionic strength sugar resistant to complement-mediated hemolysis
water that promotes the binding of complement  Cells with partial deficiency of CD59 will show
component. moderate fluorescence
 Components: TYPE III
o Sample specimen: VENOUS BLOOD or  Mutation to PIGA Mutation that causes “COMPLETE
DEFIBRINATED DEFICIENCY OF THE GPI ANCHOR” so no
o WHOLE BLOOD collected in citrate or sodium expression of CD55 and CD59.
citrate (whole blood 1:9 anticoagulant ratio)  Highly sensitive to spontaneous lysis by complement.
o Reagents: Sucrose  Cells with no CD59 added will show negative
o Ph: ~7.4 o Incubated at 370C in a low ionic fluorescence
strength sugar water solution o Promotes the  NOTE: That the absence of binding on the
binding of the complement components. granulocyte or monocytes will indicate GPI
 Procedure: Deficiency.
o Pipet 4.5mL Sucrose into each test tube (2). DIFFERENTIAL DIAGNOSIS
o Add 0.5mL of controlled blood and patient blood
 Laboratory test should demonstrate “IDIOPATHIC
on respective test tube, gently mix
INTRAVASCULAR HEMYOLSIS with or without
o Water bath in 370C for 30 minutes, centrifuge
hemoglobinuria,
o Observe the supernatant for hemolysis.
 Correlation to Ham’s Acidified test (Because it is the
 Interpretation:
confirmatory).
o Positive: Hemolysis – Possibly indicates PNH TREATMENT AND MANAGEMENT
o Negative: No Hemolysis
o Ham’s Acidified Serum Test (Old Confirmatory  Bone Marrow transplantation,
test)  Hormone Therapy,
HAM ACIDIFIED SERUM TEST  Oral iron therapy
 Eculizumab – for Classic PNH
 Principle: Erythrocyte in PNH is abnormally sensitive RED BLOOD CELL ENZYMOPATHIES
to Complement mediated lysis in acidified serum.  Genetic disorders affecting genes encoding RBC
 Components:
enzymes, causing hereditary nonspherocytic
o Sample specimen: DEFIBRINATED WHOLE hemolytic anemia
BLOOD

PADAYHAG, RIGIDOR S. | MLS-3D 10

 HEMATLOGY 2
Clinical Hematology
Midterm Topic 5- Anemia of Increased Destruction:
Intrinsic Defects
 Deficiencies in RBC enzymes that impair RBCs
functions and decrease their life span
 Main Function: Oxygen Transport over 80-120 days
 Requires functional enzymes that participate in RBC
metabolic pathways
 TWO RBC METABOLIC PATHWAYS:
o 1. Embden-Meyerhof Pathway
 a) Anaerobic pathway
 b) Generates 90% of energy in RBC
metabolism
 c) Function: ATP Production, 2-3 DPG
Formation d) Most common enzymopathy:
PYRUVATE KINASE DEFICIENCY
o 2. Hexose-Monophosphate Shunt
 a) Aerobic pathway
 b) Generates 10% of energy
 c) Function: Production of NADPH and
reduced glutathione
 Most common enzymopathy: GLUCOSE-6-
PHOSPHATE DEHYDROGENASE
DEFICIENCY
 RBCs’ capacity to detoxify is critical to maintain their
life span.
 RBCs normally produce free oxygen radicals and
hydrogen peroxide (H2O2)  They are subjected to
oxidative stress
 Glucose-6-phosphate dehydrogenase
o One of the most important intracellular
enzymes needed to protect RBC from oxidative
denaturation
o Converts glucose-6-phosphate to
phosphogluconate
o Provides the only means of generating NADPH
GLUCOSE-6-PHOSPHATE DEHYDROGENASE
(G6PD) DEFICIENCY
ETIOLOGY
 Most common RBC enyzmopathy
 An X-linked inherited disease
PADAYHAG, RIGIDOR S. | MLS-3D
RMT
2022
 Defects in G6PD gene (located on the X
chromosome)
o Men- can have normal alleles (hemizygotes) or
mutant alleles (deficient hemizygotes)
o Women- can have both alleles normal, both
mutated alleles (deficient homozygotes) or
compound heterozygotes (one normal allele
and one mutant allele
o Compound Heterozygotes- due to the random
inactivation of one of the X chromosomes in
each cell (lyonization)
o Mosaic RBCs - some having normal G6PD
activity and some cells having deficient G6PD
activity
 Has the highest prevalence in geographic areas
where malaria is endemic
 Persons who has G6PD A- variant confers protection
against Plasmodium falcipparum only in hemizygous
men
 Persons who has G6PD Mediterranean variant
confers protection against Plasmodium vivax only in
hemizygous men Reasons
 Due to parasite susceptiblity to excess free oxygen
radicals produced in G6PD-deficient RBCs
 Significant oxidative damage to the cells after
invasion in which these cells are readily phagocytized
in the reticuloendothelial system with the elimination
of the parasite
6-
11
 HEMATLOGY 2
Clinical Hematology
RMT
Midterm Topic 5- Anemia of Increased Destruction:
Intrinsic Defects
2022
 Major Contributor to the hemolytic process  Usually do not have hemoglobinuria -- indicates
 Hemoglobin (Hb) is oxidized to methemoglobin (iron extravascular hemolysis
is in the ferric state/Fe3+)
o Formation of sulfhydryl groups & disulfide
bridges -> decreased Hb solubility
 Heme is liberated from globin
 Globin denatures, forming Heinz bodies
o Adhere to the RBC membrane causing
irreversible membrane damage (cell rigidity) 
Rapidly removed by intravascular hemolysis
CLINICAL MANIFESTATIONS/PRESENTATIONS
ACUTE HEMOLYTIC ANEMIA
 Hemolysis occurs more commonly with infections,
exposure to drugs and rarely after ingestion of fava
beans (favism)
 Infections
o Most common cause of hemolysis in G6PD-
deficient individuals
o Hemoglobin can drop 3-4 g/dL if reticulocyte
production is suppressed
 Exposure to drugs
o Hemolysis secondary to drug exposure is the
classic manifestation of G6PD deficiency
o Drugs: Primaquine, Naphthalene,
Phenylhydrazine, Nitrofurantoin, Methylene
Blue
o Hemolysis begin within hours or occur
gradually 1- 3 days after intake
o Symptoms: chills, fever, headache, nausea,
back pain
o Hemoglobinuria- indicates intravascular
hemolysis − Increased reticulocytes (within 4-6
days)
o Persons with Class II and III G6PD deficiency
are clinically and hematologically normal until
the drug is taken.
 Favism
o Rare, severe hemolytic episode
o Sudden onset of acute intravascular hemolysis
− within hours after ingestion of fava beans
o Patient factors may affect the severity (type of
mutation, presence of underlying disorders,
amount of ingested fava beans)
 G6PD Mediterranean variant is commonly
associated
NEONATAL HYPERBILIRUBINEMIA
 Jaundice- 2-3 days after birth without concomitant
anemia
 Inefficient conjugation of indirect bilirubin
o Infants who have mutated G6PD gene have
homozygous mutation in the promoter region of
the bilirubin-uridine diphosphoglucuronate
glucuronosyltransferase (UGT1A1) gene
CHRONIC HEREDITARY NONSPHEROCYTIC
HEMOLYTIC ANEMIA
 Small percentage of G6PD-deficient individuals 
 Persistent hyperbilirubinemia
PADAYHAG, RIGIDOR S. | MLS-3D 12
 HEMATLOGY 2
Clinical Hematology
RMT
Midterm Topic 5- Anemia of Increased Destruction:
Intrinsic Defects
2022
 Accumulates in PK deficiency as a result of the block
in glycolysis.
 Cellular concentration of 2,3-DPG may exceed 2 or 3
times normal.
 Shift to the right  Oxygen is released to the tissues
to compensate for anemia.
CLINICAL PRESENTATION
 Varies from severe neonatal anemia and
hyperbilirubinemia to fully compensated hemolytic
process.
 Disease is usually detected in infancy or early
TREATMENT childhood
 1. Avoid exposure to potential sources of oxidant  Occasionally not detected until adulthood.
stress  Characterized by: Chronic hemolysis
 2. RBC transfusions  Manifestations:
PYRUVATE KINASE DEFICIENCY o Anemia
 Rare autosomal recessive disorder o Jaundice
 Most common form of hereditary nonspherocytic o Splenomegaly
hemolytic anemia o Gallstones (due to excessive bilirubin
 Most common enzyme deficiency of the E-M pathway production)
DEMOGRAPHICS o Folate deficiency (due to accelerated
 Most cases are found in people of Northern European erythropoiesis)
origin. o Bone marrow aplasia (usually due to parvovirus
 Most people with hemolytic anemia are doubly B19 infection)
heterozygous for two mutant genes o Skin ulcers
o In normal state, PK plays an important role in  Increased risk of iron overload and organ damage
the production of ATP, pyruvate, and lactate. that occurs with age
 ATP functions: LABORATORY FINDINGS AND CORELATIONS
o Maintains membrane shape and deformability. COMPLETE BLOOD COUNT
o Provides energy for active transport of cations.  Hgb = 6 to 12 g/dL
o Assists in modulating the amount of 2,3- DPG  Normochromic, Normocytic (MCV may be elevated
generated when reticulocytosis is marked)
ETIOLOGY AND PATHOPHYSIOLOGY  Before splenectomy, reticulocytes count = 2.5% to
 Mutation in the PKLR gene 15%
o Expressed in the liver and RBCs, where it  Following splenectomy = 56% (NOT NORMAL)
provides instructions for producing an enzyme  WBC and platelet count = NORMAL PERIPHERAL
called pyruvate kinase. BLOOD FILM
 Pyruvate kinase catalyzes the conversion of  Polychromasia, poikilocytosis, anisocytosis
phosphoenolpyruvate (PEP) to pyruvate, forming  Reticulocytes increase 5-fold
ATP. CHEMISTRY
o Marked reduction in ATP production  Serum unconjugated bilirubin = MODERATELY
o K+ and water are lost from the cell  cell ELEVATED
shrinkage, distortion of shape, and spiculation  Haptoglobin = DECREASED
o Irreversible membrane injury resulting from  Urinary urobilinogen = INCREASED
potassium loss  2,3-DPG and PEP = INCREASED
o Increased membrane calcium causes  ATP, Lactate, Pyruvate = DECREASED
decreased deformability and premature SPECIAL HEMATOLOGIC TESTS
destruction in the spleen.  Osmotic fragility test = NORMAL
 Metabolic consequences of PK deficiency:  Incubated fragility test = ABNORMAL
o ATP depletion  Autohemolyisis = VARIABLE
o Increased 2,3-DPG
 Direct antiglobulin test = NEGATIVE
RETICULOCYTES SPECIAL TEST
 Have an inordinate susceptibility to hemolysis FLUORESCENT SPOT TEST & QUANTITATIVE PK
 As mitochondria are lost during maturation, ASSAY
reticulocytes are very vulnerable to the effects of their  PK fluorescent spot test is the recommended
glycolytic defect and are destroyed in the spleen. screening test for PK deficiency.
2,3-DPG
PADAYHAG, RIGIDOR S. | MLS-3D 13
 HEMATLOGY 2
Clinical Hematology
Midterm Topic 5- Anemia of Increased Destruction:
Intrinsic Defects
 Confirmatory test: Quantitative PK assay
 PRINCIPLE | Fluorescent Spot Test Reaction
mixture, incubated with the patient hemolysate, is
added to filter paper and observed for fluorescence.
Loss of fluorescence of NADH under ultraviolet light
indicates PK activity.
o PK-deficient erythrocytes fail to complete this
reaction, and fluorescence persists for more
than 60 minutes.
 PRINCIPLE | Quantitative PK Assay Hemolysate is
prepared from patient’s anticoagulated blood after
removal of the WBCs. The change in absorbance of
the reaction mixture is measured
spectrophotometrically at 340 nm.
 Reagents:
o Phosphoenolpyruvate (PEP)
o Adenosine diphosphate (ADP)
o Lactate dehydrogenase
o Reduced form of nicotinamide adenine
dinucleotide (NADH)
SPECIMEN REQUIREMENTS
 Whole blood anticoagulated with EDTA, heparin, or
acid-citrate-dextrose (ACD) solution.
 REFERENCE RANGE
o Reported in units (U) per gram of Hb.
o Most deficient people have 5% to 25% of the
normal mean activity.
SOURCES OF ERROR
 Leukocytes contain 300 times as much PK as red
cells  must be completely removed
 Recent transfusion  provides normal cells that may
obscure PK deficiency
MUTATION DETECTION
 Accomplished by sequencing exons, flanking regions,
and promoter region of the PKLR gene.
 Increased sensitivity and specificity
 Applicable for use in prenatal testing
AUTOHEMOLYSIS TEST
 Used to screen for PK deficiency and G6PD
deficiency.
 PRINCIPLE Measures the spontaneous lysis of red
cells incubated at 37C for 48 hours. Depletion of
glucose and ATP results in membrane loss and
spherocyte formation.
SPECIMEN REQUIREMENTS
 Sterile, defibrinated blood
GENERAL PROCEDURE
 Defibrinated blood from the patient and a control are
incubated alone and with glucose (or ATP) for 48
hours.
QUALITY CONTROL
 Normal specimen must be run in duplicate  for
comparison and correct interpretation. REFERENCE
RANGE
 Normal red cells exhibit 0.2% to 2.0% hemolysis
PADAYHAG, RIGIDOR S. | MLS-3D
RMT
2022
 With glucose, 0% to 0.9% hemolysis With ATP, 0% to
0.8% hemolysis
DIFFERENTIAL DIAGNOSIS PATTERNS OF
HEMOLYSIS
TYPE I:
 Autohemolysis is slightly to moderately increased but
is partially corrected by glucose.
o Seen in G6PD deficiency, hereditary
elliptocytosis, and unstable hemoglobin
disease.
TYPE II:
 Autohemolysis is greatly increased and glucose has
no effect; however, ATP corrects the hemolysis.
o Associated with PK deficiency
HEREDITARY SPHEROCYTOSIS:
 Autohemolysis is greatly increased but can be
corrected with either glucose or ATP.
o Also found in triose phosphate isomerase
deficiency
TREATMENT AND MANAGEMENT
 Supportive treatment
 RBC transfusion
 Exchange transfusion – to prevent neonatal
hyperbilirubinemia
 Splenectomy – in severe cases
o Hemoglobin increases by 1 to 2 g/dL
o Reticulocyte counts are markedly increased
o Echinocytes are more prevalent
 Hematopoietic stem cell transplant – in children with
severe hemolytic disease
“Our greatest weakness lies in giving up. The
most certain way to succeed is always to try just
one more time.”
– Thomas A. Edison
“If you don’t go after what you want, you’ll never
have it. If you don’t ask, the answer is always no.
If you don’t step forward, you’re always in the
same place.”
– Nora Roberts
REFERENCES
University of the Immaculate Conception Powerpoint Presentation and
Discussion Prepared By Group 3 : Plaza, Braza, Dalisay, Gonzales, Guiamad,
Mayuga, Ojendras and Suniel
| MLS 3D
Keohane, E. M., Smith, L. J., & Walenga, J. M. (2020). Rodak's hematology:
Clinical principles and applications (5th ed.). St. Louis, MO: Elsevier.
Stiene-Martin, E. A., Lotspeich-Steininger, C. A., &amp; Koepke, J. A. (1998).
Clinical hematology: Principles, procedures, correlations. J.B.
Lippincott Co.: Philadelphia, Pa.
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