William Sherlock Scott Holmes BSMT-3A increase in the mean cell hemoglobin concentration (MCHC) to between 35 and

38mg/dL
CHAPTER 23: Intrinsic Defects Leading to Increased Erythrocyte Destruction increase in the red cell disribution width (RDW) to greater than 14%
SPECIAL TESTS
Intrinsic disorders can be divided into abnormalities of the
Osmotic fragility demonstrates increased RBC fragility in blood samples in which
RBC membrane, metabolic enzymes, or hemoglobin (Hb).
cells have decreased surface areatovolume ratios.
Deformability - the ability to repeatedly bend, stretch, distort, and then return to the normal
Normal biconcave RBCs show initial hemolysis at 0.45% NaCl and
Hemolysis is complete at 0.30% NaCl
discoid, biconcave shape.
curve is shifted to the left the patients RBCs have increased osmotic
The cellular properties that enable RBC deformability include:
fragility.
the RBCs biconcave, discoid geometry
curve is shifted to the right the RBCs have decreased osmotic fragility.
the viscoelastic nature (elasticity) of its membrane tail -
its cytoplasmic viscosity The osmotic fragility test is a useful confirmatory test for HS; however, a
Vertical structural integrity to the cell by anchoring the lipid bilayer to the underlying major drawback is its lack of sensitivity and specificity.
spectrin skeleton: Autohemolysis test - When normal RBCs are incubated in their own plasma,
Ankyrin complex hemolysis gradually takes place
Protein 4.1 junctional complex Normal samples less than 5.0% hemolysis
Horizontal mechanical stability, which prevents the membrane from fragmenting in In HS 10% to 50% hemolysis
response to mechanical stress: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
-spectrin can be used to identify membrane protein deficiencies by electrophoretic separation
-spectrin of the various proteins in solubilized RBC membranes with quantitation of the
Hereditary membrane defects: (see table 23-1, p. 317) proteins by densitometry.
Mutations that alter membrane structures: Radioimmunoassay membranes proteins can also be quantitated
Hereditary spherocytosis Osmotic gradient ektacytometry Variation in membrane surface area and cell
Hereditary elliptocytosis water content can be determined.
Hereditary pyropoikilocytosis (subtypeof hereditary of elliptocytosis) Flow cytometry Patient blood can be screened for HS after incubation with
Hereditary ovalocytosis,or Southeast Asian ovalocytosis eosin-5-maleimide (EMA) and measurement of the fluorescence in a flow
Mutations that alter membrane transport proteins: cytometer.
Overhydrated hereditary stomatocytosis, Hypertonic cryohemolysis test is based on the fact that cells from HS patients
Dehydrated hereditary stomatocytosis are particularly sensitive to cooling at 0 C in hypertonic solutions.
Polymerase chain reaction followed by single-strand conformational
HEREDITARY SPHEROCYTOSIS (HS) polymorphism analysis can identify potential regions in HS genes that may contain a
mutation.
Is a heterogenous group of hemolytic anemias caused by defects in proteins that disrupt the COMPLICATIONS
vertical interactions between transmembrane proteins and the underlying protein Hemolytic crises are rare and usually associated with viral syndromes.
cytoskeleton. Aplastic crises there is a dramatic decrease in hemoglobin level and reticulocyte
Mutations of six genes: count.
Anchoring proteins: Megaloblastic crisis - Patients with moderate and severe HS can also develop
-Spectrin (SPTA1) folic acid deficiency resulting from increased folate utilization to support the
-Spectrin (SPTB) chronic erythroid hyperplasia in the bone marrow.
Transmembrane proteins: TREATMENT Splenectomy
Band 3 (SLC4A1) DDX - Direct Antiglobulin test (DAT) negative
Rh-associated protein (RHAG)
Skeletal proteins: HEREDITARY ELLIPTOCYTOSIS (HE)
Ankyrin (ANK1)
Protein 4.2 (EPB42) is a heterogenous group of hemolytic anemias caused by defects in proteins that disrupt the
CLINICAL AND LABORATORY FINDINGS: horizontal or lateral interactions in the protein cytoskeleton.
Spherocytes (hallmark) and polychromasia Mutations of 3 genes:
Anemia, jaundince, splenomegaly SPTA1 codes for -spectrin
Acanthocytes some -spectrin mutations SPTB codes for -spectrin
Pincered or mushroom-shaped cells in some cases of band 3 deficiency in EPB4 codes for protein 4.1R
patients without splenectomy. CLINCAL AND LABORATORY FINDINGS
Ovalostomatocytes in homozygous EPB4.2 mutations. Few to 100% elliptocytes(cigar-shaped) and Schistocytes in severe cases
Stomatocytes in those with no expression of RHAG (the Rh-null phenotype) Asymptomatic 90% cases ; moderate to severe anemia 10% cases

HEREDITARY PYROPOIKILOCYTOSIS (HPP)

 Severe form of HE, Severe defect in spectrin that disrupts horizontal linkages in protein elevated MCHC and decreased osmotic fragility
skeleton; severe RBC fragmentation stomatocytes, target cells, burr cells
Mutation of -Spectrin (SPTA1) RBCs in which the hemoglobin appears to be puddled in discrete areas on the cell
CLINICAL AND LABORATORY FINDINGS periphery.
extreme poikilocytosis with fragmentation, microspherocytosis, and elliptocytosis NO TREATMENT
The mean cell volume (MCV) is very low (50 to 75 fL) because of the RBC
fragments. RH DEFICIENCY SYNDROME
Show marked thermal sensitivity: Fragmented @ 41-45oC; NORMAL RBCs
fragmented @ 49oC a group of rare hereditary conditions in which the expression of Rh membrane proteins
TESTS for HPP and HE TREATMENT Spelenectomy is absent (Rh-null) or decreased (Rh-mod)
Flow cytometry EMA Divided into:
SDS-PAGE amorph type (caused by mutations in Rh proteins)
regulatory type (caused by mutations in a protein that regulates Rh gene
HEREDITARY OVALOCYTOSIS OR SOUTHEAST ASIAN OVALOCYTOSIS (SAO) expression)

is a 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 (P.falciparum)
SAO is the result of one mutation, a deletion of 27 base pairs in SLC4A1, the gene that ACQUIRED STOMATOCYTOSIS
codes for band 3 In normal individuals, 3% to 5% of RBCs may be stomatocytes.
TESTS
Acute alcoholism and a wide variety of other conditions and medications have been
Ektacytometry
associated with acquired stomatocytosis.
Micropippete aspiration
CLINICAL FINDINGS - 30% oval RBCs with one to two transverse bars or ridges SPUR CELL ANEMIA

OVERHYDRATED HEREDITARY STOMATOCYTOSIS (OHST) Patients with severe liver disease develop a hemolytic anemia with acanthocytosis

A rare hemolytic anemia due to a defect in membrane cation permeability that cause the NEUROACANTHOCYTOSIS
RBCs to be overhydrated.
RBC membrane is excessively permeable to sodium and potassium at 37 C A term used to describe a group of rare inherited disorders characterized by neurologic
Mutations in the RHAG gene that codes for RhAG protein impairment and acanthocytes on the peripheral blood film.
Deficiency of stomatin, a transmembrane protein. 3 DISORDERS:
CLINCAL AND LABORATORY FINDINGS Abetalipoproteinemia (ABL) is a rare autosomal recessive disorder
Moderate to severe hemolytic anemia characterized by fat malabsorption, progressive ataxia, neuropathy, retinitis
Stomatocytes pigmentosa, and acanthocytosis.
Macrocytes (MCV of 110 to 150 fL) - ABL is caused by mutations in the MTP (microsomal triglyceride transfer
Reticulocytosis protein) gene; MTP is needed to transfer and assemble lipids onto apolipoprotein B
Reduced erythrocyte potassium concentration McLeod syndrome (MLS) is an X-linked disorder caused by mutations in the KX
Elevated erythrocyte sodium concentration gene.
KX codes for the Kx protein, a membrane precursor of the Kell blood group antigens.
Increased net cation content in the erythrocytes.
Chorea acanthocytosis (ChAc) is a rare autosomal recessive disorder
Increased osmotic fragility
characterized by chorea, hyperkinesia, cognitive impairments, and
TREATMENT Splenectomy
neuropsychiatric symptoms.
DEHYDRATED HEREDITARY STOMATOCYTOSIS (DHST) OR HEREDITARY XEROCYTOSIS -ChAc is caused by mutations in VPS13A, a gene that codes for chorein.

is an autosomal dominant hemolytic anemia due to a defect in membrane cation PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH)
permeability that causes the RBCs to be dehydrated A RARE CHRONIC INTRAVASCULAR HEMOLYTIC ANEMIA caused by an acquired clonal stem cell
Xerocyte dehydrated RBC
mutation that results in circulating blood cells that lack glycosylphosphatidylinositol
RBC membrane is excessively permeable to potassium (GPI)anchored proteins, such as CD55 and CD59.
16q23-24 gene deficiency A mutation on PIGA gene codes for a glycosyltransferase needed for the first step in the
CLINICAL AND LABORATORY FINDINGS biosynthesis of the GPI anchor to add N-acetylglucosamine to PI.
Mild to moderate anemia two GPI-anchored proteins are absent or deficient on the RBC membrane:
Reticulocytosis Decay-accelerating factor (DAF, or CD55) inhibits the formation and
Jaundice stability of the alternate pathway complement C3 and C5 convertases
Mild to moderate splenomegaly Membrane inhibitor of reactive lysis (MIRL, or CD59) prevents the formation
Pseudohyperkalemia of the membrane attack complex
Fetal loss, hydrops fetalis, and neonatal hepatitis
 RBC PHENOTYPE CLASSIFICATIONS:
Type I RBCs are phenotypically normal, express normal amounts of CD55
and CD59, and undergo little or no complement-mediated hemolysis.
Type II RBCs are the result of a PIGA mutation that causes only a partial
deficiency of CD55 and CD59, and these cells are relatively resistant to
complement-mediated hemolysis.
Type III RBCs are the result of a PIGA mutation that causes a complete deficiency
of GPI, and therefore no CD55 and CD59 proteins are anchored to the RBC
surface.
CLINICAL MANIFESTATIONS
hemolytic anemia, thrombophilia, and bone marrow failure
Hepatic vein thrombosis (Budd-Chiari syndrome) is the most common
manifestation of thrombophilia
LABORATORY FINDINGS
hemoglobinemia, hemoglobinuria, decreased level of serum haptoglobin,
increased levels of serum indirect bilirubin and lactate dehydrogenase, and
hemosiderinuria.
Reticulocyte counts are mildly to moderately increased, with less elevation.
MCV may be slightly elevated, which reflects reticulocytosis.
Osmotic fragility is normal, and the DAT result is negative.
iron deficiency develops and the RBCs become microcytic and hypochromic
Folate deficiency and Pancytopenia
The bone marrow may be normocellular to hypercellular with erythroid hyperplasia
in response to the hemolysis, or it may be hypocellular in concomitant bone
marrow failure.
Flow cytometry - deficiency of at least two GPI-anchored proteins on
granulocytes, such as CD59, CD55, CD24, CD15, is a sensitive and reproducible
method for diagnosis of PNH.
Fluorescein-labeled proaerolysin variant (FLAER) binds to the glycan core
of the GPI anchor with a high signal-to-noise ratio.
CLASSIFICATION (see page 328, table 23-3)
Classic PNH there is clinical and biochemical evidence of intravascular
hemolysis, reticulocytosis, a cellular bone marrow with erythroid hyperplasia and
normal morphology, and a normal karyotype.
PNH in the setting of another bone marrow disorder, patients have
evidence of hemolysis, but have a history of or concomitant aplastic anemia,
myeloproliferative disorder, or other myelopathy.
Subclinical PNH - patients have no clinical or biochemical evidence of
hemolysis but have a small subpopulation of GPI-deficient neutrophils that
comprise less than 1% of the total circulating neutrophils.
TREATMENT eculizumab
RED BLOOD CELL ENZYMOPATHIES
The most important metabolic pathways are:
Embden-Meyerhof pathway (anaerobic glycolysis)
hexose monophosphate (pentose) shunt
GLUCOSE-6-PHOSPHATE DEHYDROGENASE DEFICIENCY
PYRUVATE KINASE DEFICIENCY
A major function of glucose-6-phosphate dehydrogenase (G6PD) is to keep
hemoglobin iron in the reduced, physiologically active (ferrous) state for oxygen transport
and to protect hemoglobin from oxidative damage.
G6PD CLASSIFICATION
Class I includes rare variants with severe enzyme deficiency that give rise to
chronic nonspherocytic hemolytic anemia (CNSHA).
Class II includes variants with severe deficiency (less than 10% enzyme activity)
and no CNSHA.
Class III includes variants with moderate deficiency (10% to 60% enzyme
activity) and no CNSHA
Class IV includes variants with normal enzyme activity (60% to 150%)
associated with no hemolysis
Class V includes variants with increased enzyme activity (greater than 150%)
G6PD-deficient RBCs cannot generate sufficient NADPH to reduce glutathione, and
thus cannot effectively detoxify the hydrogen peroxide produced upon exposure to
oxidative stress.
CLINICAL MANIFESTATIONS
Acute Hemolytic Anemia Oxidative stress can precipitate a hemolytic
episode, and there are three main triggers:
Certain drugs anti-malarial drugs
Infections the most common cause of hemolysis in individuals with
G6PD
Fava beans Favism is a severe hemolytic episode that occurs in a small
percentage of G6PD-deficient individuals after exposure to the fava bean
Neonatal Hyperbilirubinemia mutation in the promoter region of the bilirubin-
uridine diphosphoglucuronate glucuronosyltransferase 1 (UGT1A1) gene,
which impairs their ability to conjugate and excrete indirect bilirubin.
Chronic Nonspherocytic Hemolytic Anemia RBC morphology is
unremarkable and is referred to as nonspherocytic
LABORATORY FINDINGS
The morphology of G6PD-deficient RBCs is normal except during a hemolytic
episode.
Anisocytosis, poikilocytosis, spherocytosis, and schistocytosis may occur
Bite cells (RBCs in which the margin appears indented, purportedly due to
splenic removal of a Heinz body) may be observed.
Heinz bodies
The reticulocyte count is increased and may reach 30% of RBC
Consistent with intravascular hemolysis
serum haptoglobin level is severely decreased, and there is hemoglobinemia and
hemoglobinuria.
Unconjugated (indirect) bilirubin level is also elevated.
The white blood cell (WBC) count is moderately elevated, and the platelet count
varies.
Quantitative assays of G6PD enzyme activity in RBCs can be performed to determine
the severity of the G6PD deficiency, but screening tests are usually adequate. Both
tests are based on the reduction of an oxidized pyridine nucleotide (NADP to NADPH)
during the reaction.
The principle of the screening test is the same except that, rather than
measuring the absorbance by the reduced NADPH, visual observation of the
fluorescence of reduced nucleotide when activated with ultraviolet light is used to
evaluate whether pyridine nucleotide (NADP) has been reduced.
TREATMENT - preventing the common manifestations of hemolytic anemia and neonatal
jaundice.
Pyruvate kinase (PK) is a rate-limiting key enzyme of the glycolytic pathway of RBCs. It
catalyzes the conversion of phosphoenolpyruvate to pyruvate, forming ATP.
PK deficiency are ATP depletion and an increase in 2,3-bisphosphoglycerate (2,3-BPG)
 The increase in 2,3-BPG shifts the hemoglobin-oxygen dissociation curve to the right and decreased serum haptoglobin level, and increased urinary urobilinogen.
decreases the oxygen affinity of hemoglobin The osmotic fragility of fresh cells is usually normal,
CLINICAL MANIFESTATIONS DAT result is negative.
chronic hemolysis The enzyme can be measured by spectrophotometric assay
anemia, jaundice, splenomegaly, and increased incidence of gallstones TREATMENT Splenectomy
folate deficiency
bone marrow aplasia Other Enzymopathies
skin ulcers
Iron overload hexokinase, glucose phosphate isomerase, phosphofructokinase hexokinase, glucose
LABORATORY FINDINGS phosphate isomerase, phosphofructokinase
Deficiencies of 2,3-BPG mutase and 2,3-BPG PHOSPHATASE
Reticulocytosis
increase in the number of circulating reticulocytes
Anisocytosis, poikilocytosis, and polychromasia.
burr cells, or echinocytes
The post-splenectomy peripheral blood film may also show Howell-Jolly bodies,
Pappenheimer bodies, and target cells.
platelet counts are normal or slightly increased
chronic hemolysis
increased serum indirect bilirubin level