Anemia due to

Increased Destruction of
Erythrocytes
KLIENSTAR CIARA MAGBOO, RMT, MLS (ASCPI)
Overview
•Hemolysis or hemolytic disorders refers to increased rate of
destruction (lysis) of RBCs, shortening their life span.
•The reduced number of cells results in reduced tissue
oxygenation and increased erythropoietin production by the
kidneys.
•The hemolytic process is present without anemia if bone
marrow is able to compensate by accelerating RBC
production sufficiently to replace RBCs lost through
hemolysis.
Hemolytic anemias
The cause of membrane alterations can be divided into:
A. Acute vs. Chronic
1. Acute hemolysis: has a rapid onset and is isolated (sudden),
episodic, or paroxysmal
2. Chronic hemolysis: may not be evident if the bone marrow is
able to compensate, but it may be punctuated over time with
hemolytic crises that cause the anemia
Hemolytic anemias
•The cause of membrane alterations can be divided
into:
B. Inherited vs Acquired
1. Inherited hemolytic disorders (intrinsic hemolytic
anemia)
2. Acquired hemolytic disorders in which a factor outside
the erythrocyte acts on it (extrinsic hemolytic anemia)
Hemolytic anemias
•The cause of membrane alterations can be divided into:
C. Intravascular vs Extravascular
1. intravascular hemolytic conditions: destruction of the RBCs due
to a defect within the RBCs themselves
2. Extrinsic hemolytic conditions: arise from outside the RBC,
typically substances in the plasma or conditions affecting the
anatomy of the circulatory system
Classification of Hemolytic anemia
based on site of hemolysis:
• Intravascular hemolysis:
occurs by fragmentation
Takes place most often within the bloodstream, RBCs can lyse by
fragmentation in the spleen and bone marrow
•Macrophage- mediated hemolysis:
Occurs when RBCs are engulfed by macrophages and lysed inside
the phagocyte by their digestive enzymes.
Excessive macrophage mediated
(Extravascular) hemolysis
•When an RBC is ingested by a macrophage, it is lysed within
a phagolysosome, and the contents are processed entirely
within the macrophage.
•The contents of the RBC are not detected in plasma
because it is lysed inside the macrophage, and the contents
are degraded there, hence extravascular hemolysis.
•The total plasma bilirubin level rises as RBCs are lysed
prematurely
Excessive Fragmentation
(Intravascular) Hemolysis
•It is characterized by the appearance in the plasma
of the contents of RBCs, chiefly hemoglobin, and thus
the development of (met) hemoglobinemia.
•As a result, the iron salvage proteins form complexes
with their respective ligands.
Clinical features:
1. Fatigue, dyspnea, dizziness
2. Signs of pallor and tachycardia
3. Jaundice
4. Splenomegaly, gallstones
5. Bone deformities in children
Specimen Result Fragmentation hemolysis Macrophage mediated
hemolysis
Serum Total bilirubin ↑ ↑
Indirect bilirubin ↑ ↑
LDH ↑ ↑
Haptoglobin ↓ ↓
Free hemoglobin ↑ ↑
Hemopexin ↓ ↓
Plasma Color Coffee-brown
Urine Urobilinogen ↑ ↑
Free hemoglobin + -
Methemoglobin + -
Prussian blue staining of + -
urine sediment
Urine color Root beer/beer-colored
Anticoagulated whole blood Hgb, Hct, RBC count ↓ ↓
Schistocytes +
Spherocytes +
Glycated hemoglobin ↓ ↓
Special tests Endogenous carbon ↑ ↑
monoxide
Erythrocyte life span ↓ ↓
Intrinsic Defects
Intrinsic hemolytic anemia
•Defects in the red blood cells result in premature hemolysis
and anemia
•Can be divided into:
Abnormalities of the RBC membrane
Metabolic enzymes
hemoglobin
RBC membrane structure and
Function
•In their lifespan, RBCs must repeatedly maneuver through very narrow capillaries and squeeze
through the splenic sieve
•The biconcave, discoid geometry of the RBC is dependent on vertical and horizontal interactions
between transmembrane and cytoskeleton proteins
• 2 transmembrane protein complexes that provides vertical structure integrity that prevents loss of
membrane:
Ankyrin complex
Actin

•Horizontal mechanical stability: prevents the membrane from fragmenting in response to

mechanical stress
Hereditary RBC
Membrane Abnormalities
Mutations that alter membrane
structure
Hereditary spherocytosis
It is a heterogenous group of hemolytic anemias caused by defects
in proteins that disrupts the vertical interactions between
transmembrane proteins and the underlying protein cytoskeleton
The defects in vertical membrane protein interaction cause RBCs to
lose unsupported lipid membrane over time because of local
disconnections of the lipid bilayer an underlying cytoskeleton
Mutations that alter membrane
structure
Hereditary spherocytosis
from gene mutations in which the defective proteins disrupt the
vertical linkages between the lipid bilayer and the cytoskeletal
network.
Mutation can occur in gene for:
1. ANK1: codes for ankyrin
2. SPTA1: alpha-spectrin
3. EPB42: protein 4.2
Mutations that alter membrane
structure
Hereditary spherocytosis
the defects in vertical membrane protein interactions cause the
RBCs to lose unsupported lipid membrane overtime because of local
disconnections of the lipid bilayer and underlying cytoskeleton
They have abnormal permeability to cations, particularly sodium
and potassium, which is likely a result of disruption of the integrity of
the protein cytoskeleton.
Hallmark: spherocytes
Mutations that alter membrane
structure
Hereditary spherocytosis
OFT:
Demonstrates increased RBC fragility in blood specimens in which the
RBCs have decreased surface area-to-volume ratios
Incubation: 37⁰C for 24 hours before performing
Fresh heparinized blood without hemolysis; stable for 2 hours at RT or
6 hours if the specimen is refrigerated
Mutations that alter membrane
structure
Hereditary spherocytosis
Eosin 5’-maleimide binding test:
More sensitive alternative for confirmation of HS.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
 Used to identify protein deficiencies by electrophoretic separation of
the various protein in solubilized RBC membranes with quantification
of the proteins by densitometry.
Mutations that alter membrane
structure
Hereditary spherocytosis:
3 crises:
1. Hemolytic: rare and usually associated with viral syndromes
2. Aplastic: with dramatic decrease in hemoglobin level and reticulocyte count
 Occurs in conjunction with parvovirus B19 infection that suppresses erythropoiesis, and
patient can become rapidly and severely anemic often requires transfusion.

3. Megaloblastic: can develop folate deficiency resulting from increased

utilization to support the chronic erythroid hyperplasia in the bone marrow.
Howell-Jolly bodies, target cells and Pappenheimer bodies with Leukocytosis
and thrombocytosis
Mutations that alter membrane
structure
Hereditary elliptocytosis
Defects in proteins that disrupt the horizontal or lateral interactions in the protein
cytoskeleton
Mutations on gene that disrupts the horizontal linkages:
◦ EPB41: involves protein band 4.1
◦ SPTB: involves Beta spectrin
◦ SPTA1: involves Alpha spectrin

 Loss of mechanical stability in the membrane

Polarization of cholesterol at the ends of the cell resulting to elliptocytes formation
 Most persons are asymptomatic; normal RBC lifespan
Mutations that alter membrane
structure
SOUTHEAST ASIAN OVALOCYTOSIS
 Only known mutation in Band 3
Excessive membrane rigidity

HEREDITARY PYROPOIKILOCYTOSIS
 Subtype of Hereditary elliptocytosis
 RBC fragment at 45 degrees C
◦ Normal RBC fragment at 49 degrees C
◦ Severe RBC fragmentation
 Presence of: Schistocytes, Spherocytes, Microspherocytes/ Pyropoikilocytes, Elliptocytes
Mutations that alter membrane
structure
HEREDITARY STOMATOCYTOSIS
● Cation imbalance
● Problem in Na+/K+ ATPase pump
● Increase permeability to Na and K
● Mutation: Rh-associated glycoprotein
◦ Rh null → results in stomatocyte formation
● Overhydrated: Hereditary Hydrocytosis
◦ Due to a defect in membrane cation permeability that causes
RBCs to be overhydrated
◦ ↑ Membrane permeability to sodium and potassium at 37⁰C
◦ Mutations in RHAG gene
◦ High intracellular sodium causes influx of water
◦ ↑ MCV ↓ MCHC (↓ cytoplasmic viscosity)
◦ 5-50% stomatocytes
● Dehydrated: Hereditary Xerocytosis
◦ Due to a defect in membrane cation permeability that causes
RBCs to be overhydrated
◦ Severe ↑ Membrane permeability to potassium
◦ High intracellular potassium, causes loss of water from cell
◦ ↓ MCV ↑ MCHC (↑ cytoplasmic viscosity)
◦ <10% stomatocytes
◦ Mutations in PIEZO 1 gene
 Other Hereditary membrane defects
I. Familial Pseudohyperkalemia: excessive potassium leaks out of the RBCs at room
temperature in vivo.
 Mutations in the ABCB6 gene
• Encodes a mitochondrial porphyrin transporter

II. Cryohydrocytosis: manifests as mild to moderate hemolytic anemia with stomatocytosis

caused by cold-induced leakage of sodium and potassium from the RBCs.
 Mutations in SLC4A1
• Encodes band 3 which cause it to leak cations out of the RBCs
 Mutations in GLUT1
• Encodes a glucose transporter

III. Rh Deficiency Syndrome: Rh null or decreased (Rh-mod); presents mild to moderate

hemolytic anemia with stomatocytes and occasional spherocytes
Other hereditary membrane defects
with Acanthocytosis
•Neuroacanthocytosis
It is a term used to describe a group of rare inherited disorders characterized
by neurologic impairement and acanthocytes on the PBS
I. Abetalipoproteinemia: characterized by fat malabsorption, progressive
ataxia, neuropathy, retinitis pigmentosa and acanthocytosis
II. McLeod syndrome: an X-linked disorder caused by mutations in the KX gene
III. Chorea acanthocytosis: characterized by chorea, hyperkinesia, cognitive
impairements, and neurophsychiatric symptoms
Acquired red blood cell membrane
Abnormalities
•Acquired Stomatocytosis
May occur as a drying artifact on Wright stained peripheral blood films.
In healthy individuals, 3-5% of RBCs may be stomatocytes
Acute alcoholism, medications, malignancies and CVD

•Spur Cell anemia

A small percentage of patients with severe liver disease develop a hemolytic anemia with
acanthocytosis
Free cholesterol preferentially accumulates in the outer leaflet of the RBC membrane. The
spleen remodels the membrane into acanthocytes shape
Has poor diagnosis
Acquired red blood cell membrane
Abnormalities
•PAROXYSMAL NOCTURNAL Absence of glycosylphosphatidylinositol (GPI) linked
HEMOGLOBINURIA proteins
o CD55
Intrinsic Acquired infection  Negative (-)
 DAF (Decay Accelerating Factor)

Acquired membrane defect o CD59

 Negative (-)
Increased sensitivity of red cells for  MIRL (Membrane Inhibitor of Reactive Lysis)

complement binding CD55 and CD59

o Complement inhibitory proteins
Complement mediated lysis o Important to inhibit complement
o RBCs become sensitive to lysis by o Absence can make the RBCs sensitive to lysis by
complements complement
o Both are negative in flow cytometry
 Mutation in the PIGA or PIG-A gene
 Chronic intravascular hemolysis, Hemoglobinuria,
o Code for Phosphatidylinositol Glycan Hemosiderinuria at an acid pH at nigh
Anchor Biosynthesis class A
Acquired red blood cell membrane
Abnormalities
PAROXYSMAL CONFIRMATORY TEST
• Ham’s Acidified Serum Test
NOCTURNAL • Add weak acid 0.2N HCl

HEMOGLOBINURIA • Positive Result: (+) Hemolysis

• No longer performed at present, instead we use flow
SCREENING TEST cytometry

• Sugar Water Test or Sucrose

FLOW CYTOMETRY
Hemolysis test • Currently performing flow cytometry
• Fresh blood, done within 2 hours • CD55 (-)
o Negative for DAF
o Use Citrated blood
• CD59 (-)
• Positive Result: (+) Hemolysis o Negative for MIRL
Enzyme deficiencies
Enzyme deficiencies
GLUCOSE-6-PHOSPHATE DEHYDROGENASE DEFICIENCY (G6PD)
•RBCs are particularly vulnerable to oxidative damage ad subsequent hemolysis during oxidant
stress
•G6PD is one of the important intracellular enzymes needed to protect hemoglobin and other
cellular proteins and lipids from oxidative denaturation; G6PD catalyzes the first step in a series
of reactions that detoxify hydrogen peroxide formed from oxygen radicals
•Is the most common RBC enzyme defects
•Confers protection against life-threatening P. falciparum and P. vivax
•Laboratory diagnosis:
Ascorbate cyanide screening test: detects deficiencies in the pentose phosphate pathway
Enzyme deficiencies
GLUCOSE-6-PHOSPHATE DEHYDROGENASE DEFICIENCY (G6PD)
•Enzyme deficiency in the hexose monophosphate shunt
o HMP is prevents the oxidation of hemoglobin and protein

•↓ NADPH (reduced form) ↓ glutathione

•Oxidation of hemoglobin to methemoglobin (Fe3+)
• Denatures hemoglobin – heinz bodies: stained by supravital stain
o Pitted by splenic macrophage – bite cells/blister cells

• Episodic condition, not anemic until oxidatively challenged

o Fava beans, naphthalene/moth balls, primaquine, sulfa drugs
o Can result to severe hemolytic anemia
Enzyme deficiencies
PYRUVATE KINASE DEFICIENCY
•Most common enzyme deficiency of hereditary nonspherocytic hemolytic anemia
•It is due to mutation in the PKLR gene
• Enzyme deficiency in the Embden-Meyerhof pathway
•Metabolic consequence: depletion of ATP and an increase in 2,3-BPG
•Decreased RBC deformability
•Severe hemolytic anemia
•Presence of echinocytes/burr cells (also seen in uremia)
Immune Hemolytic anemias
IgM mediated IgG mediated

Extravascular hemolysis • Clearance of C3b-sensitized • Formation of spherocytes by

RBC by macrophages mainly partial phagocytosis of IgG
in liver synthesized RBCs
• Clearance of IgG- and C3b-
sensitized RBCs by
macrophages in spleen and
liver
Intravascular hemolysis • Full IgM activation of classical • Full IgG activation of classical
complement pathway complement pathway
• Direct RBC lysis • Direct RBC lysis
Immune Hemolytic anemias
I. Warm autoimmune hemolytic
anemia
 Is the most commonly encountered
autoimmune hemolytic anemia
 The autoantibodies react optimally at
37⁰C
 Onset is usually insidious, with
symptoms of anemia
 Hemolysis is predominantly
extravascular by hepatic macrophages
 Polychromasia and spherocytes are the
typical findings on the peripheral blood
film
IgG; RBC are coated with IgG
• hagocytized by splenic macrophages
May result to membrane loss
Spherocytes:
• MCHC > 36g/dL (36 - 38 g/dL): Hyperchromic
• ↑OFT due to increased surface area
• Shift to the left
(+) DAT to differentiate from hereditary
spherocytosis
 May be:
• Idiopathic
• Secondary to diseases: CLL, lymphoma
 Immune Hemolytic anemias
II. Cold agglutinin disease/COLD AUTOIMMUNE
HEMOLYTIC ANEMIA
Usually anti-I or anti-i
Cold agglutinins are of IgM class that reacts optimally at 4⁰C and Idiopathic
are commonly found in healthy individuals

Pathologic cold agglutinins can react at body temperature  Secondary to infection:

Hemolysis is predominantly extravascular by hepatic macrophages • Anti-I: Mycoplasma pneumonia

Symptoms include acrocyanosis (a bluish discoloration of the o Caused by primary atypical

extremities)
pneumonia (PAP)
(+) DAT detects complement-coated RBCs
• Anti-i: Infectious mononucleosis
 ↑ MCHC: >38 g/dL (%)
• Increased T lymphocyte
↑ MCV

 ↓ RBC: Because automation counts based on cell size so it will no

• In tuberculosis there is increased
longer be counted as RBCs monocytes
Sample must be warmed to 37 celsius to obtain accurate RBC and
indices results RBC clumping or agglutination
Immune Hemolytic anemias
III. Paroxysmal cold hemoglobinuria
Is due to a biphasic IgG autoantibody with an  Experience hemolysis after exposure to cold
anti-P specificity
 May be:
At cold temperatures, the antibody binds to • Idiopathic
the P antigen on RBCs and partially activates • Secondary to viral infections and other
complement malignancies (e.g. measles and mumps), non-
Hodgkin lymphoma
Full complement activation and hemolysis
occur only upon warming to 37⁰C  (+) DAT
Biphasic antibody: Donath-Landsteiner  Donath Landsteiner test (+)
Antibody
Anti-P
 4 Celsius: Fixes complement to RBC
Immune Hemolytic anemias
IV. Drug Induced immune hemolytic anemia
Is suspected when there is a sudden decrease in hemoglobin after
administration of a drug, clinical and biochemical evidence of
extravascular or intravascular hemolysis, and a positive DAT results
Unifying theory: proposes that a drug interacts with the RBC
membrane and generates multiple immunogenic epitopes that can
elicit an immune response to the drug alone, to the drug-RBC
membrane protein complex, or to an RBC membrane protein alone
Thank you!