Pathology RBC and Bleeding Disorders

AY 2018-2019 Grig Misiona, MD

4th Shifting Exam 3/16/19

OUTLINE
I. Anemias
A. Anemia of Blood Loss
i. Acute Blood Loss
ii. Chronic Blood Loss
B. Hemolytic Anemias
i. Hereditary Spherocytosis
ii. G6PD Deficiency
iii. Sickle Cell Disease
iv. Thalassemia
Syndromes
v. Paroxysmal Nocturnal
Hemoglobinuria
vi. Trauma to Red Cells
C. Anemias of Diminished
Erythropoiesis
i. Megaloblastic Anemias
ii. Anemia of Folate
Deficiency
iii. Iron Deficiency Anemia
iv. Anemia of Chronic
Disease
v. Aplastic Anemia
vi. Pure Red Cell Aplasia
vii. Other Forms of Marrow
Failure
II. Polycythemia
III. Bleeding Disorders:
Hemorrhagic Diatheses
A. Caused by Vessel Wall
Abnormalities
B. Related to Reduced
Platelet Number
i. Chronic ITP
ii. Acute ITP
iii. Drug-Induced
Thrombocytopenia
iv. HIV-Associated
Thrombocytopenia
• Most common red cell indices
→ Mean cell volume (MCV)
▪ The average volume of a red cell expressed in femtoliters
(fL)
→ Mean cell hemoglobin (MCH)
▪ The average content (mass) of hemoglobin per red cell,
expressed in picograms
→ Mean cell hemoglobin concentration (MCHC)
▪ The average concentration of hemoglobin in a given
volume of packed red cells, expressed in grams per
deciliter
→ Red cell distribution width
▪ The coefficient of variation of red cell volume
Table 1. Adult reference ranges for red cells* (Robbins 9e)

TH notes: Reminder lang na Doc mentioned that most of his exam

questions would be taken from Immunohemo part of this lecture 😊

I. ANEMIAS
• ANEMIA
→ Reduction of the total circulating red cell mass below normal
limits
→ Reduces the oxygen-carrying capacity of the blood, leading
to tissue hypoxia
→ Diagnosis is based on a reduction in the hematocrit (he ratio
of packed red cells to total blood volume) and the From Robbins 9e:
hemoglobin concentration of the blood to levels that are • Whatever its cause, when sufficiently severe, anemia leads
below the normal range to certain clinical findings. Patients appear pale, and
▪ These values correlate with the red cell mass, except commonly present with weakness, malaise, and easy
when there are changes in plasma volume caused by fatigability.
fluid retention or dehydration
• The lowered oxygen content of the circulating blood leads to
• CLASSIFICATIONS OF ANEMIA dyspnea on mild exertion.
→ (1) Based on underlying mechanism (see addendum)
• Hypoxia can cause fatty change in the liver, myocardium,
→ (2) based on alterations in red cell morphology and kidney
▪ Red cell size (normocytic, microcytic, or macrocytic)
▪ Degree of hemoglobinization reflected on the color of
• MANIFESTATIONS
red cells (normochromic or hypochromic)
→ Severe fatty changes in the myocardium are sufficient to
▪ Shape
cause cardiac failure and compound tissue hypoxia due to
the deficiency of oxygen in the blood.
In general:
→ Myocardial hypoxia may manifest as angina pectoris,
• Microcytic, hypochromic anemias
particularly when complicated by pre-existing coronary artery
→ Caused by disorders of hemoglobin synthesis (most often
disease.
iron deficiency)
→ With acute blood loss and shock, oliguria and anuria may
• Macrocytic anemias
develop as a result of renal hypoperfusion.
→ Often stem from abnormalities that impair the maturation
→ CNS hypoxia can cause headache, dimness of vision and
of erythroid precursors in the bone marrow
faintness.
• Normochromic, normocytic anemias
→ Diverse etiologies; to identify cause, specific abnormalities
of red cell shape are investigated through peripheral
smears
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 → Accumulation of hemoglobin degradation products that are
A. ANEMIA OF BLOOD LOSS created as part of the process of red cell hemolysis
• The physiologic destruction of senescent red cells takes place
ACUTE BLOOD LOSS within macrophages, which are abundant in the spleen, liver,
• The effects of acute blood loss are mainly due to the loss of and bone marrow.
intravascular volume → This process is triggered by age-dependent changes in red
→ If massive, can lead to cardiovascular collapse, shock and cell surface proteins, which lead to their recognition and
death phagocytosis.
• The clinical features depend on the rate of hemorrhage and • Majority of hemolytic anemias involve the premature destruction
whether the bleeding is internal or external. of red cells within phagocytes, an event referred to as
• If the patient survives: extravascular hemolysis

Blood volume is rapidly restored by fluid shift from the interstitial • EXTRAVASCULAR HEMOLYSIS
to the intravascular fluid compartment → If persistent, extravascular hemolysis leads to a hyperplasia
↓ of phagocytes manifested by varying degrees of
Hemodilution and a lowering of the hematocrit spenomegaly.
↓ → Generally caused by alterations that render the red cell less
Reduction in oxygenation deformable.
↓ → Regardless of the cause, the principal clinical features of
Triggers the increased secretion of erythropoietin from the kidney extravascular hemolysis are:
↓ ▪ Anemia
Proliferation of committed erythroid progenitors (CFU-E) in the ▪ Splenomegaly
marrow ▪ Jaundice
↓ → Some hemoglobin inevitably escapes from phagocytes which
Maturation and release of new red cells (reticulocytes) into the leads to variable decreases in plasma haptoglobin, an α2-
peripheral blood after 5 days. globulin that binds free hemoglobin and prevents its
excretion in the urine.
NOTE: → Because pathologic destruction of red cells occurs mostly in
• The iron in hemoglobin is recaptured if red cells the spleen, individuals with extravascular hemolysis often
extravasate into tissues, whereas bleeding into the gut or benefit from splenectomy.
out of the body leads to iron loss and possible iron
deficiency, which can hamper the restoration of normal red • INTRAVASCULAR HEMOLYSIS
cell counts. → Less common
→ May be caused by:
• Significant bleeding results in changes not only in red cells but ▪ Mechanical injury
also in white cells (leukocytosis) and platelets ▪ Complement fixation
(thrombocytosis). ▪ Intracellular parasites
→ If bleeding is sufficiently massive to cause a decrease in the ▪ Exogenous toxic factors (e.g. clostridial sepsis)
blood pressure, there will be a compensatory release of → Whatever the mechanism, intravascular hemolysis is
adrenergic homones that mobilizes granulocytes from the manifested by:
intravascular margignal pool and results in leukocytosis. ▪ Anemia
→ Initially, red cells appear normal in size and color ▪ Hemoglobinemia
(normocytic, normochromic), but as marrow production − The large amounts of free hemoglobin released from
increases, there is a striking increase in reticulocyte count lysed red cells are promptly bound by haptoglobin,
(reticulocytosis). producing a copmplex that is rapidly cleared by
▪ Reticulocytes – are larger in size than normal red cells, mononuclear phagocytes.
and have a blue-red polychromatophilic cytoplasm ▪ Hemoglobinuria
• The clinical features depend on the rate of hemorrhage and − As serum haptoglobin is depleted, free hemoglobin
whether the bleeding is internal or external. oxidizes to methemoglobin, which is brown in color.
• If the patient survives: − Renal proximal tubular cells reabsorb and catabolize
much of the filtered hemoglobin and methemoglobin,
but some passes out in the urine → red brown urine
CHRONIC BLOOD LOSS
▪ Hemosiderinuria
• Induces anemia only when the rate of loss exceeds the
− Iron released from hemoglobin can accumulate within
regenerative capacity of the marrow or when iron reserves are
tubular cells and cause renal hemosiderosis
depleted, causing iron-deficiency anemia.
▪ Jaundice
− Heme groups derived from hemoglobin-haptoglobin
B. HEMOLYTIC ANEMIAS
complexes are catabolized to bilirubin within
• Common features of hemolytic anemias:
mononuclear phagocytes, leading to jaundice
→ A shortened red cell life span below the normal 120 days ▪ Unlike in extravascular hemolysis, splenomegaly is NOT
→ Elevated erythropoietin levels and a compensatory increase seen
in erythropoiesis

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• In all types of uncomplicated hemolytic anemias, excess serum
bilirubin is unconjugated.
• The level of hyperbilirubinemia depends on the functional
capacity of the liver
→ Normal liver – rarely severe jaundice
• Excessive bilirubin excreted by the liver to the GI tract leads to
the formation of gallstones derived from heme pigments.
Figure 1. Marrow smear from a patient with hemolytic anemia. The marrow
reveals increased numbers of maturing erythroid progenitors (normoblasts).
HEREDITARY SPHEROCYTOSIS (HS)
• An inherited disorder caused by intrinsic defects in the red cell
membrane skeleton that render red cells spheroid, less
deformable and vulnerable to splenic sequestration.
• Autosomal dominant inheritance pattern
• HS is caused by a diverse mutations that leat to an insufficiency
of membrane skeletal components
→ As an effect, life span of the affected cells is decreased on
average to 10 to 20 days
Figure 2. Red cell membrane skeleton in hereditary spherocytosis. Top image
shows the normal organization of the major red cell membrane skeletal
proteins.Mutations involving α-spectrin, β-spectrin, ankyrin, band 3, or band 4.2
weaken the interactions between these proteins and cause the red cells to lose
membrane fragments (bottom image) making the red cells more spherical and
less deformable than normal ones. (Robbins 9e)
Patho RBC and Bleeding Disorders
PATHOGENESIS
• Most of the mutations cause frameshift or introduce premature
stop codons, such that the mutated allele fails to produce any
protein
• The resulting deficiency of the affected protein reduces the
assembly of the skeleton as a whole, destabilizing the overlying
plasma membrane
• Young HS red cells are normal in shape. But the destabilized
lipid bilayer sheds membrane fragments as red cells age in the
circulation.
• The loss of membrane relative to cytoplasm forces the cells to
assume the smallest possible diameter for a given volume, i.e. a
sphere.
• Compound beneficial effects of splenectomy prove that the
spleen has a cardinal role in the premature demise of
spherocytes.
• Normal red cells must undergo extreme deformation to leave
the cords of Billroth and enter the sinusoids.
• Because of their spheroidal shape and reduced deformability,
the hapless spherocytes are trapped in the splenic cords, where
they are easy prey for macrophages.
• The splenic environment also somehow exacerbates the
tendency of HS red cells to lose membrane along with K+ ions
and water; prolonged splenic exposure (eryhtrostasis), depletion
of red cell glucose and diminished red cell pH
→ After splenectomy, the spherocytes persists, but the anemia
is corrected
Figure 3. Pathophysiology of hereditary spherocytosis. (Robbins 9e)
CLINICAL FEATURES
• Diagnosis is based on family history, hematologic findings and
laboratory evidence
• Red cells are abnormally sensitive to osmotic lysis when
incubated in hypotonic salt solutions, which causes the influx of
water into spherocytes with little margin for expansion.
• HS red cells also have an increased mean cell hemoglobin
concentration (MCHC), due to dehydration caused by the loss
of K+ and water.
• Characteristic features:
→ Anemia, splenomegaly, and jaundice
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• In a small minority, HS presents at birth with marked jaundice
and requires exchange transfusions.
• In 20%-30% of patients, the disease is so mild and
asymptomatic
• In most patients, the compensatory increase in erythropoiesis is
outpaced, hence producing a chronic hemolytic anemia of mild
to moderate severity
• Generally stable clinical course may sometimes be punctuated
by aplastic crises usually triggered by an acute parvovirus
infection
• Transfusions may be necessary to support patient until the
immune response clears the infection
• Hemolytic crises may also occur, and this is produced by
intercurrent events leading to increased splenic destruction of
red cells (e.g. infectious mononucleosis)
Figure 4. Peripheral blood smear showing anisocytosis and several dark-
appearing spherocytes with no central pallor. Howell-Jolly bodies (small
dark nuclear remnants) are also present in red cells of this asplenic
patients. From Robbins 9e.
GLUCOSE-6-PHOSPHATE DEHYDROGENASE
DEFICIENCY
• Deficiency or impairment in enzyme function cause
abnormalities in the hexose monophosphate shunt → reduced
ability of red cells to protect themselves against injuries and →
hemolysis
• The most important enzyme derangements is the hereditary
deficiency of glucose-6-phosphate dehydrogenase (G6PD)
activity.
• G6PD reduces nicotinamide adenine dinucleotide phosphate
(NADP) to NADPH while oxidizing glucose-6-phosphate
• NADPH then provides reducing equivalents needed for the
conversion of oxidized glutathione (GSSG) to reduced
glutathione (GSH) which protects against oxidant injury by
participating as a cofactor in reactions that neutralize compounds
such as peroxide (H2O2)
Patho RBC and Bleeding Disorders
Figure 5. Role of glucose-6-phosphate dehydrogenase (G6PD) in defense
against oxidant injury.
• G6PD deficiency is a recessive X-linked trait
• Males are at a higher risk for symptomatic disease
• Most G6PD variants are harmless, and two variants designated
G6PD- and G6PD Mediterranean cause most of the clinically
significant hemolytic anemias
• G6PD- is present in about 10% of American blacks, while G6PD
Mediterranean is prevalent in the Middle East
→ The high frequency of these variants in each population is
believed to stem from a protective effect against Plasmodium
falciparum malaria.
• The episodic hemolysis that is characteristic of G6PD deficiency
is caused by exposures that generate oxidant stress
• The most common triggers are:
→ Infections
▪ Free radicals are produced by activated leukocytes
▪ Some infections that can trigger hemolysis include viral
hepatitis, pneumonia, and typhoid fever.
→ Drugs
▪ Antimalarials (primaquine and chloroquine)
▪ Sulfonamides
▪ Nitrofurantoins
→ Food (fava beans)
• Exposure of G6PD deficient red cells to high levels of oxidants
causes the cross-linking of reactive sulfhydryl groups on globin
chains
→ Cause the denaturation and formation of membrane-bound
precipitates called Heinz bodies
• G6PD deficiency presents as neonatal jaundice or a chronic
low-grade hemolytic anemia in the absence of infection or
known environmental triggers
Figure 5. Peripheral blood smear of G6PD deficient red cells. Red cells with
precipitates of denatured globin called Heinz bodies (inset). As the splenic
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 macrophages pluck out the inclusions, “bite cells” are produced (Robbins
9e).
• Because hemolytic episodes related to G6PD deficiency occur
intermittently, features related to chronic hemolysis (e.g.
splenomegaly and cholelithiasis) are absent.
SICKLE CELL DISEASE
• A common hereditary hemoglobinopathy caused by a point
mutation in β-globin → promotes the polymerization of
deoxygenated hemoglobin → red cell distortion, hemolytic
anemia, microcytic vascular obstruction, and ischemic tissue
damage
• Hemoglobin is a tetrameric protein composed of two pairs of
globin chains (a pair of alpha, and a pair of beta chains)
• Normal adult red cells mainly contain:
→ HbA (α2β2)
→ Smaller amounts HbA2 (α2δ2)
→ Fetal hemoglobin HbF (α2γ2)
• Mutant hemoglobin substitutes for the normal β-globin to
generate HbS
• Autosomal recessive disorder
Figure 6. Pathophysiology of the sickle cell disease
• As HbS polymers frow (or as deoxygenation grows), they
herniate through the membrane skeleton and project from the
cell ensheathed by only the lipid bilayer
• This severe derangement in membrane structure causes the
influx of Ca2+ ions → induce the cross-linking of membrane
proteins and activate an ion channel that permits the efflux of K +
and water.
• With repeated episodes of sickling, red cells become
increasingy dehydrated, dense, and rigid, which retain a sickle
shape even when fully oxygenated are eventually formed.
ETIOLOGY
• Substitution of valine for glutamic acid at the sixth position of
β-globin
PATHOGENESIS
Patho RBC and Bleeding Disorders
• When deoxygenated, HbS polymerizes into long, stiff chains
that deform (sickle) RBCs, causing chronic hemolysis,
microvascular occlusion, and tissue damage
• Sickled red cells are mechanically fragile, leading to
intravascular hemolysis
• Sickled red cells are rapidy sequestered and removed by
mononuclear phagocytes (extravascular hemolysis)
• Microvascular occlusion with resultant tissue hypoxia and
infarction is the most clinically important clinical aspect of sickle
cell disease
• Propensity to occlude small vessels is dependent on:
→ (1) percentage of irreversibly sicked cells in the blood
→ (2) stickiness of sickled RBCs (sickled RBCs express
increased levels of adhesion molecules)
→ (3) Local inflammation and platelet aggregation
• Free hemoglobin released from ruptured RBCs binds and
inactivates nitric oxide (NO) – increasing vascular tone and
enhancing platelet aggregation
MORPHOLOGY
• Peripheral blood shows variable numbers of irreversibly sickled
cells, reticulocytosis, and target cells due to RBC dehydration
• Bone marrow shows normoblastic hyperplasia
→ When hyperplasia is severe, expansion of the marrow can
cause bone resorption; extramedullary hematopoiesis can
occur
CLINICAL FEATURES
• Chronic hemolytic anemia (hematocrits from 18%-30%)
• Vaso-occlusive crises present as painful episodes of ischemic
necrosis, most commonly involving bones, lungs (acute chest
syndrome), liver, brain, penis, and spleen
• Aplastic crisis
• Sequestration crises
• Progressive splenic fibrosis and impairment of the alternate
complement pathway predisposing to infections
DIAGNOSIS
• Based on
→ Clinical findings
→ Sickle cells in the peripheral blood smear
▪ Detection of HbS by hemoglobin electrophoresis
Figure 7. Irreversibly sickled cell in the center.
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 VARIABLES AFFECTING RATE AND DEGREE OF SICKLING
• Infection of HbS with other types of hemoglobin within RBCs
→ HbS constitutes only 40% of the hemoglobin with the
remainder being HbA in heterozygotes
→ HbA and other beta globins (HbF) interfere with HbS
polymerization
→ Sickle cell disease is considerably less severe with
hereditary persistence of HbF
→ HbC
▪ Another variant of hemoglobin which forms HbSC with
HbS.
▪ HbSC cell tend to lose water and salt, which increases
propensity for hemolysis and sequestration.
• Mean corpuscular hemoglobin concentration (MCHC)
→ Dehydration increases intracellular concentration of HbS
which increases probability of HbS interacting
→ 𝑴𝑪𝑯𝑪 =
𝑯𝒈𝒃
𝑯𝒄𝒕
• Intracellular pH
→ Decreased pH decreases hemoglobin oxygen affinity,
thereby increasing the proportion of deoxygenated HbS and
the propensity to polymerize
• Microvascular transit time
→ Normally, the capillary transit rate is sufficiently high that
significant deoxygenation (and therefore sickling) cannot
occur
• Sickling is usually confined to tissues with intrinsically sluggish
blood flow (e.g. spleen, bone marrow) or those involved by
inflammation, where transit rates are retarded.
THALASSEMIA SYNDROMES
• Caused by inherited mutations that decrease the synthesis of
either the α-globin or β-globin chains
→ Both chains compose HbA (adult hemoglobin; α2β2) which
leads to anemia, tissue hypoxia and red cell hemolysis
• α – 2 α-globin genes on chromosome 16 encodes both α2
→ normal chains denoted as (α/α α/α) – 4 genes total
• β – 1 β-globin gene on chromosome 11 encodes both β2
→ normal chains denoted as (β/β) – 2 genes total
• Hematologic consequences of deficient chain synthesis stems
from both chain deficiency and excess of the other globin chain
• Prevalence seems to be explained by the protection they afford
heterozygous carriers against malaria in regions where it is
endemic (Mediterranean basin, Asia, tropical Africa, etc)
• Two mechanisms which cause anemia:
→ ↓ RBC production
→ ↓ Red cell lifespan
β-Thalassemias
• Caused by mutations that diminish the synthesis of β-globin
chains
MOLECULAR PATHOGENESIS
• Two categories of mutations which cause β-thalassemia:
→ β0 mutations, associated with absent β-globin synthesis
→ β+ mutations, characterized by reduced (but detectable) β-
globin synthesis
1. Splicing Mutations
• Most common cause of β+-thalassemia
• Most mutations lie within introns, although a few are located
within exons
Patho RBC and Bleeding Disorders
• Some of these mutations destroy normal RNA splice junction →
completely prevents production of normal β-globin mRNA → β0-
thalassemia
• Others create ectopic splice site within introns → flanking
normal splice sites remain → both normal and abnormal splicing
occurs and some normal β-globin mRNA is made → β+-
thalassemia
2. Promoter Region Mutations
• Reduces transcription by 75-80%
• Some normal β-globin is synthesized → β+-thalassemia
3. Chain Termination Mutations
• Most common cause of β0-thalassemia
• Two subtypes of mutations fall into this category, both blocking
translation and preventing synthesis of functional β-globin:
→ Most common type creates a new stop codon within an exon
→ The other introduces small insertions or deletions that shift
the mRNA reading frames (frameshift mutations)
Figure 8. Sites of gene mutations causing β-thalassemia.
MORPHOLOGY
• Microscopic appearance: hypochromic, microcytic red cells
• RBCs are underhemoglobinized with subnormal oxygen
transport capacity
• Peripheral lood smears show anisocytosis and poikilocytosis
• Codocytes / target cells (so called because hemoglobin
collects in the center of the cell), basophilic stippling, and
fragmented red cells are also common
• Inclusions of aggregated α chains are efficiently removed by the
spleen and not easily seen
• Reticulocyte count is ↑, but it is lower than expected for the
severity of anemia because of ineffective erythropoiesis
• Variable numbers of normoblasts are seen in PBS due to
“stress” erythropoiesis and abnormal release from sites of
extramedullary hematopoiesis
• Other major alterations:
→ Bone Marrow
▪ In the untransfused patient there is a striking expansion of
hematopoietically active marrow
▪ In bones of the face and skull the burgeoning marrow
erodes existing cortical bone and induces new bone
formation, giving rise to a ”crewcut” appearance on x-ray
→ Spleen
▪ Both phagocyte hyperplasia and extramedullary
hematopoiesis contribute to enlargement of the spleen,
which can weigh as much as 1500 g
→ Liver and Lymph Nodes
▪ Enlarged by extramedullary hematopoiesis
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Figure 9. Crewcut appearance due to new bone growth.
CLINICAL FEATURES
• Imbalance in α- and β-globin synthesis → diminished survival of
RBCs and their precursors
• Unpaired α chains precipitate within red cell precursors →
formation of insoluble inclusions → damages RBCs (i.e.
membrane damage – proximal cause of RBC pathology) →
apoptosis
→ In severe β-thalassemia: 70% to 85% of red cell precursors
suffer this fate → ineffective erythropoiesis
→ Those red cells that are released from the marrow also
contain inclusions and have membrane damage, leaving
them prone to splenic sequestration and extravascular
hemolysis
• Ineffective erythropoiesis → massive erythroid hyperplasia in
the marrow and extensive extramedullary hematopoiesis (in
liver, spleen, lymph nodes) → erosion of the bony cortex →
impairment of bone growth → skeletal abnormalities
• Ineffective erythropoiesis → suppression of hepcidin (negative
regulator of iron) → ↑absorption of dietary iron and repeated
blood transfusions → severe iron accumulation →
hemosiderosis and secondary hemochromatosis → deposit
on and damage heart, liver, and pancreas
CLINICAL SYNDROMES
• Based on the genetic defect β+ or β0 and the gene dosage
β -THALASSEMIA MAJOR
• Individuals with two β-thalassemia alleles (β+/β+, β+/β0, or β0/β0)
• Severe-transfusion dependent anemia
• Most common in Mediterranean countries, parts of Africa and
Southeast Asia
• Anemia manifests 6 to 9 months after birth as hemoglobin
synthesis switches from HbF to HbA
• If untransfused, hemoglobin reaches a level of 3-6 g/dL
• Red cells may completely lack HbA (β0/β0 genotype) or contain
small amounts (β+/β+ or β0/β+ genotypes)
• Markedly ↑ HbF levels (major Hb present here)
• HbA2 levels are sometimes high but more often are normal / low
• Growth retardation and death can occur at an early age due to
profound anemia
• Blood transfusions can lessen the anemia and suppress bone
deformities
→ However, in multiple transfusions, patiens can often die d/t
cardiac failure resulting from progressive iron overload and
secondary hemochromatosis
• Cheekbones and other bony prominences are enlarged and
distorted
• Hepatosplenomegaly d/t extramedullary hematopoiesis
Patho RBC and Bleeding Disorders
• Tx: Hematopoietic stem cell transplantation is the only
therapy offering a cure
Figure 10. Pathogenesis of -thalassemia major. Note that aggregates of
unpaired -globin chains, a hallmark of the disease, are not visible in routinely
stained blood smears. Blood transfusions are a double-edged sword, diminishing
the anemia and its attent complications, but also adding to the systemic iron
overload.
β -THALASSEMIA MINOR
• Much more common than β-thalassemia major
• Heterozygotes with one β-thalassemia gene and one normal
gene (β+/β or β0/β) usually are asymptomatic or have mild
microcytic anemia
• HbA2 elevated to 4-8% from 2.5% (elevated ratio of δ-chain to
β-chain synthesis)
• HbF generally normal or slightly increased
• In PBS:
→ Hypochromia, microcytosis, basophilic stippling, target cells
• Important to differentiate from IDA due to superficial
resemblance and need for genetic counseling
→ Differentiate using tests for serum iron, TIBC, and ferritin
→ Increase in HbA2 is diagnostic for β-thalassemia minor
β -THALASSEMIA INTERMEDIA
• includes milder variants of (β+/β+) or (β+/β0)-thalassemia and
unusual forms of heterozygous β-thalassemia
• 2 types:
→ Those who have two defective β-globin genes and an α-
thalassemia gene defect, which improves the effectiveness
of erythropoiesis and red cell survival by lessening the
imbalance in α- and β-chain synthesis
→ Those individuals have a single β-globin defect and one or
two extra copies of normal α-globin genes (stemming from a
gene duplication event), which worsens the chain imbalance
α-Thalassemias
• Caused by inherited deletions that result in reduced or absent
synthesis of α-globin chains
• Normal: 4 α-globin genes for 2 α-globin chains
• Severity depends on how many genes are affected
• Anemia stems both from a lack of adequate hemoglobin and the
presence of excess unpaired globin chains (β, γ, and δ), which
vary in type at different ages (same with β–thalassemia)
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• Hemoglobin Barts: γ4 tetramers formed by excess unpaired γ-
globin chains; seen in newborns with α-thalassemia
• HbH: β4 tetramers formed by excess β-globin chains; seen in
older children and adults with α-thalassemia
CLINICAL SYNDROMES
• Determined and classified by the number of α-globin genes that
are deleted.
• α-thalassemia syndromes stem from combinations of deletions
that remove one to four α-globin genes
SILENT CARRIER STATE
• Associated with the deletion of a single α-globin gene (α/- α/α)
→ barely detectable reduction in chain synthesis
• Asymptomatic patients but with slight microcytosis
α-THALASSEMIA TRAIT
• Caused by:
→ deletion of 2 α-globin genes from a single chromosome (-/-
α/α)
→ deletion of 1 α-globin gene from each of the two
chromosomes (α/- α/-)
• Both produce similar quantitative deficiencies of α-globin and
are clinically identical, but have different implications for the
children of affected individuals, who are at risk of clinically
significant α-thalassemia (Either HbH or Hydrops fetalis) if at
least one parent has -/-
• Identical clinical picture with β -thalassemia minor
→ Microcytosis
→ Minimal or no anemia
→ No abnormal physical signs
→ HbA2 are normal or low
HEMOGLOBIN H DISEASE
• caused by deletion of 3 α-globin genes (-/- α/-)
• most common in Asian populations
• synthesis of α chains is markedly reduced
• HbH:
→ β4 tetramers from excess β-globin chains
→ has an extremely high affinity for oxygen and therefore is not
useful for oxygen delivery, leading to tissue hypoxia
disproportionate to the level of hemoglobin
• HbH is prone to oxidation, which causes it to precipitate and
form intracellular inclusions → red cell sequestration and
phagocytosis in the spleen → moderately severe anemia
resembling β-thalassemia intermedia
HYDROPS FETALIS
• Most severe form of α-thalassemia
• Caused by deletion of all 4 α-globin genes (-/- -/-)
• Hemoglobin Barts:
→ γ4 tetramers from excess γ-globin chains
→ Too high affinity for oxygen that they deliver little to tissues
• Early fetal development is permitted by embryonic ζ-chain
synthesis
→ ζ-chains + γ-chains → functional ζ2γ2 Hb tetramer
• Signs of fetal distress usually become evident by the third
trimester of pregnancy
• Severe tissue anoxia → death in utero or shortly after birth
→ intrauterine transfusion can be life saving
Patho RBC and Bleeding Disorders
• Manifestations similar to that seen in hemolytic disease of the
newborn
→ Severe pallor
→ Generalized edema
→ Massive hepatosplenomegaly
• Lifelong dependence on blood transfusion
→ Risk of iron overload
• Hematopoietic stem cell transplantation can be curative
Figure 11. Summary of thalassemias.
PAROXYSMAL NOCTURNAL HEMOGLOBINURIA
• Disease that results from acquired mutations in the
phosphatidylinositol glycan complementation group A gene
(PIGA)
→ Enzyme essential for the synthesis of certain membrane-
associated complement regulatory proteins
• Only hemolytic anemia caused by an acquired genetic defect
Pathogenesis
• PIGA mutations lead to deficient expression of a family of
proteins normally anchored into the cell membrane via
glycosylphosphatidylinositol (GPI)
• PIGA is X-linked and subject to lyonization / random inactivation
→ Mutation occurs in hematopoietic stem cell, thus all future
cell progeny are affected
→ Normal people also have these mutations; it is theorized that
the number of PIGA-mutated cells increase in an
autoimmune reaction to combat GPI-linked antigens
▪ PNH is often associated with aplastic anemia
• Missing GPI proteins:
→ Membrane inhibitor of reactive lysis (CD59)
▪ most important
▪ potent inhibitor of C3 convertase
▪ Prevents spontaneous activation of alternative pathway
→ decay-accelerating factor (CD55)
→ C8-binding protein
• GPI-linked proteins affected regulate complement inactivation
• Deficiency leads to abnormal susceptibility to lysis or injury by
complements (intravascular hemolysis)
Clinical Features
• Chronic intravascular hemolysis without dramatic
hemoglobinuria – more typical
→ Only 25% manifest as paroxysmal and nocturnal hemolysis
• Nocturnal - tendency for red cells to lyse at night
→ Slight decrease in blood pH during sleep → increases the
activity of complement
• Anemia is mild to moderate
• Heme iron lost in urine (hemosiderinuria) → iron deficiency →
worsens anemia if left untreated
• Thrombosis
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 → Leading cause of disease-related death in individuals with
PNH
→ 40% - suffer from venous thrombosis, often involving the
hepatic, portal, or cerebral veins
→ 5-10% - develop acute myeloid leukemia or a
myelodysplastic syndrome
Diagnosis
• Flow cytometry
→ Provides a sensitive means for detecting red cells that are
deficient in GPI-linked proteins such as CD59
Treatment
• Eculizumab
→ Monoclonal antibody
→ Prevents the conversion of C5 to C5a
→ Reduces the hemolysis and attendant transfusion
requirements
→ Lowers the risk of thrombosis by up to 90%
→ Drawbacks
▪ High cost
▪ Increased risk of serious or fatal meningococcal infections
• Immunosuppressive drugs
→ Beneficial for those with bone marrow aplasia
• Hematopoietic stem cell transplantation
→ Only cure
TH: Focus on this part! Marami daw questions iaask sa part na to
😉 membrane is removed → red cell assumes spherical shape
IMMUNOHEMOLYTIC ANEMIA
• Premature destruction of red cells due to antibody binding
• Commonly referred to as autoimmune hemolytic anemias
• Classified based on the characteristics of the responsible Ab
Diagnosis
• Direct Coombs antiglobulin test (DCT or DAT)
→ Patient’s red cells are mixed with sera containing antibodies
specific for human immunoglobulin or complement
→ If either Ig or complement is present on the surface of the red
cells → antibodies cause agglutination visually appreciated
as clumping
→ Procedure:
▪ Blood is drawn and red cells are washed with NSS
(remove plasma and unbound Abs of px)
▪ Red cells are incubated with anti-human globulin (Coombs
reagent)
▪ The anti-human antibodies against human antibodies
attached to red cells causes agglutination
• Indirect Coombs antiglobulin test (IAT)
→ Patient’s serum is tested for its ability to agglutinate
commercially available red cells bearing particular defined
antigens
→ Characterize the antigen target and temperature dependence
of the responsible antibody
• Quantitative immunologic tests
→ Direct measurement of Ab
Patho RBC and Bleeding Disorders
Figure 12. Classification of immunohemolytic anemias.
WARM ANTIBODY TYPE
• Most common form of immunohemolytic anemia
• About 50% of cases are idiopathic (primary)
→ Other 50% due to drugs or predisposing condition
▪ If due to lymphoid neoplasm, transfusion is hard due to
incompatibility → administer corticosteroids
• Cause: IgG class (less commonly, IgA)
• Mostly extravascular hemolysis
→ IgG coat red cells (anti-RBC; anti-Rh in most idiopathic
cases) and act as opsonins → IgG Fc receptors attach to
phagocytes, inducing “partial” phagocytosis as red cell
→ sequestered in spleen → moderate splenomegaly due to
hyperplasia of splenic phagocytes
• Treatment: removal of inducing agent if drug; else,
immunosuppressive drugs or splenectomy
Mechanism of Drug-Induced Hemolytic Anemia
• Hemolysis occurs 1-2 weeks after large IV dose of drug is
started
• Antigenic drugs: Drugs (e.g., penicillin, cephalosporins,
quinidine) bind to the RBC surface → antibodies interact with
drug (like in penicillin) or an RBC-drug complex (like in
quinidine)
→ Intravascular hemolysis due to Abs fixing complement
→ Extravascular hemolysis due to Abs acting as opsonin for
phagocytes
• Tolerance-breaking drugs: Drugs (prototype: α-methyldopa)
induce autoantibodies against intrinsic RBC antigens, especially
Rh antigens
→ 1% develop clinically significant hemolysis
COLD AGGLUTININ TYPE
• Cause: IgM that bind red cells avidly at low temperatures (0°C
to 4°C)
• Less common than warm antibody immunohemolytic anemia
(15% to 30% of cases)
• Appear transiently after certain infections:
→ Mycoplasma pneumoniae
→ Epstein-Barr virus (EBV)
→ Cytomegalovirus (CMV)
→ Influenza virus
→ Human immunodeficiency virus (HIV)
• Self-limited and rarely induce clinically important hemolysis
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 cont. Cold Agglutinin Type − Bombay phenotype (Oh) – A, B, and H are
• Chronic cold agglutinin immunohemolytic anemia occur in: missing; thus the patient cannot be transfused with
→ Certain B-cell neoplasms blood type O
→ Idiopathic condition − H Ag amount in blood types: O > A2 > B > A2B > A1
• Clinical symptoms result from RBC agglutination and > A1B
complement fixation in vascular beds cooler than 30°C (like in → ABO typing discrepancies
tips of exposed fingers, toes, ears) ▪ Group I – weak or absent Ab reaction
• IgM binding and complement fixation is rapid but as the blood (immunosuppressed px)
recirculates and warms → IgM is released, usually before ▪ Group II – weak or absent Ag reaction (A or B
complement-mediated hemolysis can occur subgroups)
• Transient interaction of IgM is sufficient to create sublytic ▪ Group III – unexpected Ab reaction (autoantibodies)
quantities of C3b (opsonin) ▪ Group IV – unexpected Ag reaction (cold reactive
→ Minimal complement-mediated hemolysis, but the antibodies)
complement-coated cells are readily phagocytized in spleen, • Rh (or D antigen)
liver, and bone marrow → 3 alleles: C and c, E and e, and D (d denotes absence of
→ Hemolytic anemia is of variable severity; vascular obstruction D, is not an actual allele)
in areas exposed to cold temperatures results in pallor, → Autosomal dominant
cyanosis, and Raynaud phenomenon → Abs are mostly IgG, formed upon exposure to foreign Ag
• Treatment: balutan ng kumot si patient ▪ Hemolytic disease of newborn (HDN)
▪ Do not bind complement
COLD HEMOLYSIN TYPE → Tested using IAT in pregnant women
• Cause: IgG autoantibodies responsible for an unusual entity • Other major blood group systems:
known as paroxysmal cold hemoglobinuria → Lewis
→ Causes substantial, sometimes fatal, intravascular hemolysis ▪ Produced by secretions but adsorbed on RBCs
and hemoglobinuria ▪ Abs are IgM, naturally occurring
→ IgGs against the P blood group → Bind to RBCs in cool, ▪ Phenotypes:
peripheral regions of the body − Le (a-b-) – found in cord blood, cancer, pregnant
→ RBCs recirculate to warm regions → Complement-mediated − Le (a+b-) or Lea
lysis − Le (a-b+) or Leb – associated with H.pylori and
→ In other words, IgG binds when cold, then causes lysis when Norwalk virus
it warms − Lex – marker for Reed-Sternberg cells
• Most cases occur in children after viral infections and are → MNSs
transient ▪ Found only on RBC
▪ Exhibits dosage effect
Extra Immunohematology stuff (wala sa Robbins or lecture, ▪ Components:
pero sabi ni doc na 60% of questions niya from IHA so here’s − Glycophorin A – MN
some extra info): − Glycophorin B – Ss
• Most immunogenic blood types: ABO > Rh > Kell ▪ Antibodies:
• ABO Blood Group − Anti M – IgM, reacts best at pH 6.5 (acidic)
→ Has naturally occurring antibodies (blood type A has − Anti N – IgM, found in hemodialysis px
antigen A and anti-B, etc) − Anti S, Anti s - IgG
▪ IgM that react at room temperature ▪ Phenotypes:
− React best below body temp − (M-N-) or MN null – resistant to P. falciparum
− Does not cause hemolytic disease of newborn infection
(HDN) because IgM cannot cross placenta − (M+N-) – higher M amount than (M+N+)
→ Testing for ABO grouping: →P
▪ Forward/Direct typing – testing unknown Ag on ▪ Antigens
patient RBC with known commercial Ab (blue anti-A or − P – receptor for Parvovirus B19
yellow anti-B dye) − Pk – receptor for Shiga toxin and E. coli
▪ Backward/Indirect typing – testing unknown Ab in ▪ Antibodies
patient serum with known red cell suspension − Anti-P – biphasic hemolysis (bind at cold, lyse at
→ Antigens in ABO blood group: warm)
▪ A antigen – N-acetyl-D-galactosamine − Anti-P1 – weak IgM, from liver flukes
− A1 – 80% of those with blood type A → Ii
− A2 – 20%; may produce anti-A1 (so pwedeng kahit ▪ Public antigen – found in all people
blood type A tinransfuse sa A na px, may reaction) ▪ Anti-I – Mycoplasma pneumonia, cold heme agglutinin
▪ B antigen – D-galactose disease (CHAD)
▪ H antigen – L-fucose ▪ Anti-i – HEMPAS, alcoholic cirrhosis
− H antigen yung kinakabitan ng A or B sa RBC, so if → Kell
there is only H with no A or B, the blood type is “O” ▪ 3rd most immunogenic
▪ K (Kell) – private Ag
▪ k (cellano) – public Ag
Patho RBC and Bleeding Disorders 10 of
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 → Kidd C. ANEMIAS OF DIMINISHED ERYTHROPOIESIS
▪ For urea transport • Causes:
▪ Methyldopa induces Abs against Kidd (Jka) → Nutritional deficiencies (most common)
− Severe warm autoimmune hemolytic anemia → Secondary to renal failure
▪ Jka & Jkb – not easily destroyed by enzymes → Chronic inflammation
→ Duffy
▪ Chemokine receptor MEGALOBLASTIC ANEMIAS
▪ Phenotypes: • Impairment of DNA synthesis that leads to ineffective
− Fya & Fyb – destroyed by enzymes hematopoiesis and distinctive morphologic changes, including
− Fy (a-b-) – resistance to P. vivax and knowlesi abnormally large erythroid precursors and red cells
→ Lutheran • Macrocytic, normochromic anemia
▪ Anti-Lua – IgG, IgM, IgA • Two principal types:
▪ Anti-Lub – IgG → Pernicious anemia (Major form of vitamin B12 deficiency
anemia)
IgG IgM IgG & IgM → Folate deficiency anemia
Rh ABO MN
Duffy (Fy) Ii
Morphology
Kidd (Jk) P1
• Prominent peripheral blood anisocytosis with abnormally large
Kell (K) Lutheran (Lu)
and oval RBCs (macro-ovalocytes)
Ss Lewis (Le)
• May appear hyperchromic but MCHC is normal
Enzyme enhanced Enzyme destroyed Cold reacting
• Marrow: Erythroid precursor nuclear maturation lags behind
P MNS ABO
cytoplasmic maturation; ineffective erythropoiesis is reflected by
Rh Fy MN
increased apoptosis with compensatory megaloblastic
I Chido-Rogers (Ch P
hyperplasia
Jk & Rg) Lea
• Abnormal granulopoiesis with giant metamyelocytes in
Le Leb
marrow and hypersegmented neutrophils in peripheral blood

HEMOLYTIC ANEMIA RESULTING FROM TRAUMA

TO RED CELLS
• Turbulent flow and increased shear forces cause RBC
fragmentation and intravascular hemolysis; peripheral blood
reveals fragmented RBC (schistocytes), “burr cells,” “helmet
cells,” and “triangle cells”
• Causes:
→ Cardiac valve prostheses
▪ Artificial mechanical cardiac valves are more frequently
implicated than are bioprosthetic
→ Microangiopathic hemolytic anemia
▪ Microvascular lesion that results in luminal narrowing,
often due to the deposition of fibrin and platelets.
▪ Most commonly seen with disseminated intravascular
coagulation
▪ Also occurs in: thrombotic thrombocytopenic purpura
(TTP), hemolytic-uremic syndrome (HUS), malignant
hypertension, SLE, and disseminated cancer

Figure 13. Microangiopathic hemolytic anemia.

Figure 14. uses of megaloblastic anemia.

Patho RBC and Bleeding Disorders 11 of

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Figure 15. Megaloblastic anemia (bone marrow aspirate). A to C,
Megaloblasts in various stages of differentiation. Note that the orthochromatic
megaloblast (B) is hemoglobinized (as revealed by cytoplasmic color), but in
contrast to normal orthochromatic normoblasts, the nucleus is not pyknotic.
The early erythroid precursors (A and C) and the granulocytic precursors
are also large and have abnormally immature chromatin
ANEMIAS OF VIT B12 DEFICIENCY:
PERNICIOUS ANEMIA
• specific form of megaloblastic anemia caused by an
autoimmune gastritis that impairs the production of
intrinsic factor (IF), which is required for vitamin B12 uptake
from the gut
• NORMAL VITAMIN B12 METABOLISM:
• Microorganisms are the ultimate source of vitamin B12
(cobalamin); plants and vegetables contain little cobalamin, and
most dietary cobalamin comes from animal products.
• Peptic digestion releases dietary vitamin B12; it is bound to
salivary proteins called R binders.
• R-B12 complexes are digested in the duodenum by pancreatic
proteases; released vitamin B12 binds to intrinsic factor (IF), a
protein secreted by parietal cells of the gastric fundus.
• IF-B12 complexes bind to IF receptors in the distal ileum
epithelium; absorbed vitamin B12 complexes with
transcobalamin II and is transported to tissues.
• 1% of ingested vitamin B12 can be absorbed through an
alternative pathway independent of intrinsic factor or the
terminal ileum.
Figure 16. Normal B12 metabolism. (See appendix for a larger image)
Patho RBC and Bleeding Disorders
PERNICIOUS ANEMIA
• Incidence
→ Occurs in all racial group but somewhat more prevalent in
Scandinavian and other Caucasian population
→ Disease of older adults with median age of diagnosis at 60
▪ Rare in people younger than 30
→ Genetic predisposition is strongly suspected
PATHOGENESIS
• Pernicious anemia is a specific form of megaloblastic anemia
• Caused by autoimmune gastritis and attendant loss of intrinsic
factor (IF) production
• Autoreactive T cell response initiates gastric mucosal injury and
triggers the formation of autoantibodies; autoantibodies are not
the primary cause of disease butare of diagnostic utility and can
exacerbate the process:
→ Type I antibodies (present in 75% of patients) block B12
binding to IF
▪ Type I AB found in both plasma and gastric juice
→ Type II antibodies block IF or IF-B12 binding to the ileal
receptor
→ Type III antibodies (i.e., 85% to 90% of patients) directed
against parietal proton pump proteins affect acid secretion
▪ Recognize the α and β subunits of the gastric proton
pump, which is normally localized to the microvilli of the
canalicular system of the gastric parietal cell.
▪ Type III AB are not specific, in that they are found in as
many as 50% of older adults with idiopathic chronic
gastritis not associated with pernicious anemia
• When the mass of intrinsic factor-secreting cells falls below a
threshold (and reserves of stored vitamin B12 are depleted),
anemia develops.
• The common association of pernicious anemia with other
autoimmune disorders (ex: autoimmune thyroiditis and
adrenalitis) is also consistent with an underlying immune basis.
• The tendency to develop pernicious anemia (as well as other
autoimmune disorders) is linked to genetic variants involving the
inflammasome, suggesting that altered innate immunity may
also play some role.
MORPHOLOGY
• Bone marrow shows megaloblastic erythroid hyperplasia, giant
myelocytes and metamyelocytes, hypersegmented neutrophils,
and large, multilobed nuclei in megakaryocytes
• Stomach shows diffuse chronic gastritis
• Gastric fundal atrophy, affecting both chief cell and parietal
cells, with virtual absence of parietal cells
• Glandular epithelium is replaced by by mucus-secreting goblet
cells that resemble those lining the large intestine, a form of
metaplasia (“intestinalization”)
• Gastric atrophy and metaplastic changes are due to
autoimminuty and not Vit B12 deficiency
→ Hence, parenteral administration of vitamin B12 corrects the
megaloblastic changes in the marrow and the epithelial cells
of the alimentary tract, but gastric atrophy and achlorhydria
persist.
• Some of the cells as well as their nuclei may increase to double
the normal size
• Atrophic glossitis: the tongue is shiny, glazed, and red
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• Central nervous system (CNS) lesions occur in 75% of cases,
characterized by demyelination of dorsal and lateral spinal
cord tracts,sometimes followed by loss of axons
→ Give rise to spastic paraparesis, sensory ataxia, and severe
paresthesias in the lower limbs.
→ Less frequently, degenerative changes occur in the ganglia
of the posterior roots and in peripheral nerves
HISTOLOGY
• Chronic atrophic gastritis (marked by loss of parietal cells)
• Prominent infiltrate of lymphocytes and plasma cells
• Megaloblastic changes in mucosal cells similar to those founds
in erythroid precursors
CLINICAL FEATURES
• Onset is insidious, with symptoms due to anemia and
posterolateral spinal tract involvement
• Anemia is often quite severe by the time it comes to medical
attention. The course is progressive unless halted by therapy.
• Diagnosis is based on the (1) presence of moderate to severe
megaloblastic anemia, (2) leukopenia with hypersegmented
IRON DEFICIENCY ANEMIA
neutrophils/granulocytes, (3) low serum B12 levels, and (4)
• Most common nutritional disorder in the world
elevated homocysteine and methylmalonic acid as
consequences of diminished thymidine and methionine
synthesis
• The diagnosis is confirmed by profound reticulocytosis and a
rise in Hct beginning about 5 days after parenteral B12
administration
→ Serum anti-IF antibodies are highly specific for pernicious
anemia (their presence attests to the cause rather than the
presence or absence of Vit B12 deficiency)
• There is a significant association of pernicious anemia with
other autoimmune disorders of the adrenal and thyroid glands,
as well as increased risk of gastric cancer
• Persons with atrophy and metaplasia of the gastric mucosa
associated with pernicious anemia have an increased risk of
gastric carcinoma
• Elevated homocysteine levels are a risk factor for
atherosclerosis and thrombosis, and it is suspected that vitamin
B12 deficiency may increase the incidence of vascular disease
on this basis.
• With parenteral or high-dose oral vitamin B12, the anemia is
cured and the progression of the peripheral neurologic disease
can be reversed or at least halted.
→ But the changes in the gastric mucosa and the risk of
carcinoma are unaffected.
ANEMIA OF FOLATE DEFICIENCY
→ A deficiency of folic acid (more properly,
pteroylmonoglutamic acid)
→ Folate is involved in single carbon transfers in a variety of
biochemical pathways
→ Deficiency induces a megaloblastic anemia hematologically
indistinguishable from that seen with B12 deficiency
▪ Serum homocysteine levels are also increased, but
methylmalonate concentrations are normal
→ However gastric atrophy and the neurologic sequelae of B12
deficiency do not occur
→ Diagnosis of folate deficiency requires demonstration of
reduced serum or RBC folate levels
→ Deficiency occurs primarily with (1-3):
Patho RBC and Bleeding Disorders
▪ (1) Inadequate intake (e.g., chronic alcoholics, very
elderly, or indigents)
▪ (2) Increased requirements/demand (e.g., pregnancy,
infancy, derangements associated with hyperactive
hematopoiesis (hemolytic anemia) and disseminated
cancer)
▪ (3) Impaired utilization
▪ Folate antagonists (e.g., methotrexate for
chemotherapy)
→ Humans are entirely dependent on dietary sources for their
folic acid requirement, which is 50 to 200 μg daily.
→ Although prompt hematologic response heralded by
reticulocytosis follows the administration of folic acid, it
should be remembered that the hematologic symptoms of
vitamin B12 deficiency anemia also respond to folate
therapy.
→ Folate does not prevent (and may even exacerbate) the
neurologic deficits seen in vitamin B12 deficiency states.
▪ It is thus essential to exclude vitamin B12 deficiency in
megaloblastic anemia before initiating therapy with folate.
• Iron Metabolism
→ Normal Western diet contains 10 to 20 mg of iron daily,
mostly as heme iron in animal products
▪ The remainder is inorganic iron from vegetables
→ Intake is usually sufficient to balance daily losses of 1
to 2 mg from sloughed skin and gastrointestinal
epithelial cells
→ Some 15% to 20% of total body iron is in stored form
bound to hemosiderin or ferritin
▪ Serum ferritin level is a good indicator of total iron
stores
→ Major sites of iron storage: liver and mononuclear
phagocytes
→ The rest of the body’s iron is complexed in a number of
functional proteins
▪ 80% of functional iron is contained in hemoglobin
with myoglobin, catalase, and cytochromes making
up the rest
→ Excess iron can be highly toxic, so that uptake must be
carefully regulated
→ Iron balance is maintained by regulating the
absorption of dietary iron across the duodenal
epithelium
→ Heme iron enters mucosal cells directly (20% is
absorbable)
▪ Non-heme iron is first reduced to ferrous iron via
cytochome B before transport; (1% to 2% of non-
heme iron is absorbed)
→ Absorbed iron is transported across the basolateral
membrane, where it is bound to plasma transferrin for
distribution throughout the body
▪ This basolateral transport requires ferriportin, a
membrane transporter, and hephaestin to re-oxidize
the reduced iron
→ The remaining intracellular iron is bound to ferritin and
subsequently lost when the epithelium is sloughed
during normal turnover
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 → Iron homeostasis is regulated in large part by hepcidin,
a hepatic peptide that blocks duodenal iron
transepithelial transport by inducing the degradation of
ferroportin
▪ As hepcidin levels decrease (e.g., with reduced iron
stores or increased erythropoiesis), ferroportin
expression is increased, and iron transport into the
bloodstream is enhanced
→ Conversely, as stores become full, hepcidin levels
increase, ferroportin is degraded, and iron transport
into the bloodstream is blocked
→ Hepcidin also blocks the release of iron from
macrophages, an important source of iron for heme
synthesis in erythropoiesis
→ Abnormalities in hepcidin levels lead to disturbances in
iron metabolism ranging from some forms of anemia to
hemochromatosis (systemiciron overload)
(see appendix for a larger image)
ETIOLOGY
• Iron deficiency results from:
→ Lack of dietary iron
→ Impaired iron absorption
→ Increased iron requirement
→ Chronic blood loss (most common cause)
• 1 mg/day of iron needed to maintain normal iron balance
• Only 10%-15% of ingested iron is absorbed therefore daily iron
requirement is:
→ 7-10 mg of iron for adult men
→ 7-20 mg of iron for female
• Bioavailability of iron is also important
→ Heme iron is more absorbable than inorganic iron
→ Absorption of inorganic iron is enhanced by ascorbic acid,
citric acid, amino acids, and sugars
→ Absorption of inorganic iron is inhibited by tannates,
carbonates, oxalates, and phosphates
• Dietary iron inadequacy occurs in the following groups:
→ Infants – high risk due to very small amount of iron in milk
→ Impoverished – suboptimal diets for socioeconomic reasons
→ Older adults – restricted diets of meat because of limited
income or poor dentition
→ Teenagers – mainly consumes “junk” food
• Impaired absorption is found in:
→ Sprue, fat malabsorption, chronic diarrhea
→ Gastrectomy (decreased acidity and faster transit time)
• Chronic blood loss includes external hemorrhage, bleeding into
the genitourinary tracts
Patho RBC and Bleeding Disorders
• Iron deficiency in adult men and postmenopausal women is
attributed to GI blood loss until proven otherwise
PATHOGENESIS
• Iron deficiency produces a hypochromic microcytic anemia
regardless of cause
• At the outset of chronic blood loss or other states of negative
iron balance, iron reserves in the form of ferritin and
hemosiderin may be adequate to maintain normal hemoglobin
and hematocrit levels as well as normal serum iron and
transferrin saturation
• Progressive depletion of iron stores first lowers serum iron and
transferrin saturation levels without producing anemia
→ Concomitant increase erythroid activity in the bone marrow
• Anemia appears only when iron stores are completely depleted
and is accompanied by lower than normal serum iron, ferritin,
and transferrin saturation levels
MORPHOLOGY
• Mild to moderate increase in erythroid progenitors in bone
marrow
• Diagnostic finding- disappearance of stainable iron from
macrophages in the bone marrow, best assessed by
performing Prussian blue stains on smears of aspirated marrow.
• Peripheral blood smear- red cells are microcytic and
hypochromic
• Normal red cells with sufficient hemoglobin have a zone of
central pallor measuring about 1/3 of the cell diameter
• Established iron deficiency- zone of pallor is enlarged,
hemoglobin may be seen only in a narrow peripheral rim
• Poikilocytosis in the form of small, elongated red cells (pencil
cells) is also characteristically seen
CLINICAL FEATURES
• Clinical manifestations of anemia are nonspecific
• Dominating signs and symptoms relate to the underlying cause
(e.g. GI or gynecologic disease, malnutrition, pregnancy,
malabsorption)
• Severe and long-standing IDA – depletion of iron-containing
enzymes leads to
→ koilonychia, alopecia, atrophic changes in tongue and gastric
mucosa, intestinal malabsorption
→ Depletion of iron from CNS = pica and periodically move their
limbs dring sleep
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• Esophageal webs appear together with microcytic hypochromic APLASTIC ANEMIA
anemia and atrophic glossitis to complete the triad of major • A syndrome of chronic primary hematopoietic failure and
findings in the rare Plummer-Vinson syndrome attendant pancytopenia
• Diagnosis depends on laboratory studies → There is anemia, neutropenia, and thrombocytopenia
→ Decreased hemoglobin and hematocrit • Autoimmune mechanisms are suspected but inherited or
→ In association with hypochromia, microcytosis, modest acquired abnormalities of hematopoietic stem cells also
poikilocytosis contribute
→ Low serum iron and ferritin; high total plasma iron-binding • Doc Misiona: Death in aplastic anemia is usually due to
capacity (reflects elevated transferrin levels) infection
→ Low serum iron with increased iron-binding capacity results
in a reduction of transferrin saturation to below 15%.
• Reduced iron stores inhibit hepcidin synthesis, and its serum
levels fall. ETIOLOGY
• Oral iron supplementation produces increased reticulocytes
about 5-7 days followed by steady increase in blood counts and • Most cases of known etiology follow exposure to chemicals and
normalization red cell indices drugs (e.g. cancer chemotherapy drugs, benzene)
→ Drugs and agents cause marrow suppression that is dose
ANEMIA OF CHRONIC DISEASE related and reversible
• Impaired red cell production associated with chronic diseases • Other cases arise in an unpredictable, idiosyncratic fashion
that produce systemic inflammation following exposure to drugs that normally cause little or no
• There is a reduction in the proliferation of erythroid progenitors marrow suppression (e.g. chloramphenicol and gold salts)
and impaired iron utilization • Persistent marrow aplasia can also appear after a variety of
• Doc Misiona: This type of anemia is commonly seen in elderly viral infections (e.g. viral hepatitis of the non-A, non-B, non-C,
• The chronic illnesses associated with this form anemia can be non G type)
grouped into: • Whole body irradiation can destroy hematopoietic stem cells in
→ Chronic microbial infections – osteomyelitis, bacterial a dose-dependent fashion
endocarditis, lung abscess • Specific abnormalities underlie some cases of aplastic anemia
→ Chronic immune disorders – rheumatoid arthritis, regional such as:
enteritis → Fancomi anemia – rare autosomal recessive disorder
→ Neoplasms – carcinoma of lung and breast, Hodgkin caused by defects in a multiprotein complex that is required
lymphoma for DNA repair
• Anemia occurs in the setting of persistent systemic inflammation → Inherited defects in telomerase – partial deficits in
and is associated with low serum iron, reduced total iron-binding telomerase activity could result in premature hematopoietic
capacity, and abundant stored iron in tissue macrophages stem cell exhaustion and marrow aplasia
• IL-6 stimulate an increase in hepatic production of hepcidin → Abnormally short telomeres
→ Hepcidin inhibits ferroportin function in macrophages • In most instances, no initiating factor can be identified
→ Hepcidin reduces the transfer of iron from the storage pool to
developing erythroid precursors in the marrow
→ As a result, erythroid precursors are starved for iron despite
plenty of available iron
• Erythropoeitin levels are inappropriately low for this degree of
anemia
→ Studies suggests hepcidin directly or indirectly suppresses
erythropoietin production
• Iron sequestration in the setting of inflammation enhances the
body’s ability to fend off certain types of infection particularly
bacteria that requires iron for pathogenicity
→ Hepcidin is structurally related to defensins
• Anemia is usually mild with the dominant symptoms relating to
the underlying disease
• Red cells are normocytic and normochromic, or hypochromic
and microcytic
• Presence of increased storage iron in marrow macrophage,
high serum ferritin level, and reduced total iron-binding capacity
readily rule out iron deficiency as cause of the anemia
• Successful treatment of the underlying condition reliably
corrects the anemia
→ Some patients, particularly those with cancer, benefit from
administration of erythropoietin
Figure 19. Major causes of aplastic anemia.

Patho RBC and Bleeding Disorders 15 of

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 PATHOGENESIS
• Two major etiologies have been invoked:
→ Extrinsic immune-mediated suppression of marrow
progenitors
→ Intrinsic abnormality of stem cells
• Activated T cells suppress hematopoietic stem cells
→ Stem cells may first be antigenically altered by exposure to
drugs, infectious agents, or other environmental insults
→ This provokes a cellular immune response resulting to the
release of interferon-γ and TNF by TH1 cells resulting to the
suppression and killing of hematopoietic progenitors
• Aplastic anemia can also result from a fundamental stem cell
abnormality
→ Occasional transformation of aplasias into myeloid
neoplasms, typically myelodysplasia or acute myeloid
leukemia; and the association with abnormally short
telomeres
→ Some marrow insults presumably result in sufficient injury to
limit proliferative and differentiation capacity of stem cells

Figure 21. Aplastic anemia.

CLINICAL FEATURES
• Can occur at any age and in either sex with usually insidious
onset
• Initial manifestations vary but pancytopenia ultimately appears
→ Anemia causes progressive weakness, pallor, and dyspnea
→ Thrombocytopenia is heralded by petechiae and ecchymosis
→ Neutropenia manifests as frequent and persistent minor
infections or sudden onset of chills, fever, and prostration
• Splenomegaly is characteristically absent
• Red cells are usually slightly macrocytic and normochromic
→ Reticulocytopenia is the rule
• Diagnosis rests on examination if a bone marrow biopsy
• In aplastic anemia, the marrow is hypocellular (and usually
markedly so), whereas myeloid neoplasms are associated with
hypercellular marrows filled with neoplastic progenitors.
• Prognosis is variable
→ Bone marrow transplantation is the treatment of choice in
those with a suitable donor
• Older patients or those without suitable donors often respond
MORPHOLOGY
well to immunosuppressive therapy
• Markedly hypocellular bone marrow largely devoid of
hematopoietic cells
→ Only fat cells, fibrous stroma, and scattered lymphocytes and PURE RED CELL APLASIA
plasma cells remain
• Primary marrow disorder (likely autoimmune)
• Marrow aspirates often yield little material, hence aplasia is best
→ Only erythroid progenitors (RBC precursors) are
appreciated in marrow biopsies
suppressed (sometimes completely absent)
• Nonspecific pathological changes are related to
• Associated with:
granulocytopenia and thrombocytopenia such as
→ Neoplasms (thymoma and large granular lymphocytic
mucocutaneous bacterial infections and abnormal bleeding
leukemia)
• Systemic hemosiderosis can appear if the anemia necessitates
→ Drug exposures
multiple transfusions
→ Autoimmune disorders
→ Parvovirus infection
• Management:
→ Resection- for px with thymoma
→ Immunosupressive therapy – for px without thymoma

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 → Plasmapheresis - px with pathogenic autoantibodies, such as ▪ Eg. Dehydration, prolonged vomiting, diarrhea, excessive
neutralizing antibodies to erythropoietin use of diuretics.
→ Due to Stress polycythemia
• Autoimmune basis except for Parvovirus B19 (the virus itself kills ▪ “Gaisböck syndrome”
the RBC precursor cells) ▪ An obscure condition of unknown cause
• Course of patients infected by Parvovirus B19: • Absolute polycythemia:
→ Immunocompetent individuals: Transient red cell aplasia (clears → When there is an increase in total red cell mass
the viral infection in 1 to 2 weeks ) → Primary:
→ Px with hemolytic anemias: Aplastic crisis ▪ Increase in RBC mass is due to an erythropoietin-
→ Immunocompromised individuals: Chronic red cell aplasia independent manner
▪ Results from an intrinsic abnormality of hematopoietic
precursors
OTHER FORMS OF MARROW FAILURE ▪ Eg. Polycythemia vera
MYELOPHTHISIC ANEMIA o Most common cause
o Myeloproliferative disorder associated with
• Cause of Anemia: Space-occupying lesions replace normal mutations that lead to erythropoietin-independent
marrow elements growth of red cell progenitors
• Lesions: metastatic cancer (breast, lung, prostate) or any → Secondary:
infiltrative process involving the marrow ▪ Increase in RBC mass is due to increased erythropoietin
→ Causes marrow distortion and fibrosis ▪ May be compensatory or pathologic
• Depresses productive capacity of the bone marrow by ▪ Eg. Erythropoietin-secreting tumors and inherited
→ 1. Replacing the normal marrow elements with fibrosis defects that lead to the stabilization of HIF-1α (hypoxia-
→ 2. Disturbing the RBC and granulocytes release from the induced factor that stimulates the transcription of the
marrow erythropoietin gene)
▪ Leukoerythroblastosis :produced due to the abnormal
release from marrow
▪ Teardrop-shaped red cells: deformed during their
tortuous escape from the fibrotic marrow

CHRONIC RENAL FAILURE

• Cause of Anemia (in renal failure): due to the diminished
synthesis of erythropoietin by the damage kidneys which
leads to decreased red cell production
• Associated with anemia that is proportional to the severity of the
uremia
• Management
→ Administration of Recombinant Erythropoietin
→ Iron replacement therapy (gives optimal response)

HEPATOCELLULAR LIVER DISEASE

• Cause of Anemia: due to bone marrow failure often
exacerbated by folate and iron deficiencies caused by poor
nutrition and excessive bleeding
• Associated with anemia (slightly macrocytic) whether if it is
Figure 22. Pathophysiologic classification of polycythemia.
toxic, infectious, or cirrhotic in origin
• Erythroid progenitors are mainly affected but white cells and
platelets may be depressed in some cases III. BLEEDING DISORDERS:
HEMORRHAGIC DIASTHESES
ENDOCRINE DISORDERS • Excessive bleeding can result from
• Hypothyroidism may be associated with mild normochromic, → increased fragility of vessels
normocytic anemia → platelet deficiency or dysfunction
→ derangement of coagulation, alone or in combination

II. POLYCYTHEMIA TEST TO EVALUATE HEMOSTASIS

• Abnormally high red cell count (usually with increased Prothrombin Time (PT)
hemoglobin level) • Extrinsic and common coagulation pathways
• Relative polycythemia: • Clotting of plasma (after addition of an exogenous source of
→ When there is hemoconcentration due to decreased tissue thromboplastin and Ca2+ ions) is measured in seconds
plasma volume • Causes of prolonged PT:

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 → deficiency or dysfunction of factor V, factor VII, factor X,
prothrombin, or fibrinogen
Partial Thromboplastin Time (PTT)
• Intrinsic and common coagulation pathways
• The clotting of plasma (after addition of kaolin, cephalin, and
Ca2+ ions) is measured in seconds
• Causes of Prolonged of the PTT:
→ can be due to deficiency or dysfunction of factors V, VIII, IX,
X, XI, or XII, prothrombin, or fibrinogen, or to interfering
antibodies to phospholipid
Platelet Count
• Obtained on anticoagulated blood using an electronic particle
counter
• Reference range is 150-300 × 103 platelets/μL
• Abnormal platelet counts are best confirmed by inspection of a
peripheral blood smear
• Clumping of platelets can cause spurious “thrombocytopenia”
during automated counting
• High counts may be indicative of a myeloproliferative disorder,
such as essential thrombocythemia
Test of Platelet Function
• Specialized tests that can be useful in particular clinical settings
→ Tests of platelet aggregation, which measure the ability of
platelets to aggregate in response to agonists like thrombin
→ Quantitative and Qualitative tests of von Willebrand
factor
▪ Plays an important role in platelet adhesion to the
extracellular matrix
• Bleeding time, an old test, has some value but is time-
consuming and difficult to standardize and is therefore
performed infrequently
A. BLEEDING DISORDERS CAUSED BY VESSEL WALL
ABNORMALITIES
• Disorders of increased vascular fragility are common but do not
usually cause serious bleeding problems
• Clinical Presentation:
→ Small hemorrhages (petechiae and purpura)
→ Occasionally, hemorrhages into joints, muscles, and
subperiosteal locations
→ Menorrhagia, nosebleeds, gastrointestinal bleeding, or
hematuria
Clinical conditions in which abnormalities in the vessel wall
cause bleeding:
INFECTIONS
• Presentation: petechial and purpuric hemorrhages
→ Eg. Meningococcemia, other forms of septicemia, infective
endocarditis, and several of the rickettsioses
• MOA: Microbial damage to the microvasculature (vasculitis) and
disseminated intravascular coagulation
DRUG REACTIONS
• Presentation: cutaneous petechiae and purpura without
causing thrombocytopenia
Patho RBC and Bleeding Disorders
• MOA: Deposition of drug-induced immune complexes in vessel
walls, which leads to hypersensitivity (leukocytoclastic)
vasculitis
SCURVY AND EHLERS-DANLOS SYNDROME
• MOA: Microvascular bleeding that results from collagen defects
that weaken vessel walls
• Cushing syndrome: Poor vascular support due to loss of
perivascular supporting tissue
→ MOA: Protein-wasting effects of excessive corticosteroid
production cause loss of perivascular supporting tissue
HENOCH-SCHONLEIN PURPURA
• Systemic immune disorder of unknown cause
• Presentation: purpuric rash, colicky abdominal pain,
polyarthralgia, and acute glomerulonephritis
• MOA: Deposition of circulating immune complexes within
vessels throughout the body and within the glomerular
mesangial regions
HEREDITARY HEMORRHAGIC TELANGIECTASIA
(WEBER-OSLER-RENDU SYNDROME)
• Autosomal dominant disorder
• MOA: that can be caused by mutations in at least five different
genes, most of which modulate TGF-β signaling
• Presentation: Dilated, tortuous blood vessels with thin walls
that bleed readily anywhere,
→ Bleeding on mucous membranes of the nose (epistaxis),
tongue, mouth, and eyes, and throughout the gastrointestinal
tract
PERIVASCULAR AMYLOIDOSIS
• MOA: Weaken blood vessel walls causing bleeding
• Most common with amyloid light-chain (AL) amyloidosis and
often manifests as mucocutaneous petechiae
•
B. BLEEDING RELATED TO REDUCED PLATELET
NUMBER: THROMBOCYTOPENIA
THROMBOCYTOPENIA
• Reduction in platelet number
• Important cause of generalized bleeding
• Defined as platelet count less than 100,000 platelets/Ul
→ 20,000-50,000 platelets/uL: may aggravate posttraumatic
bleeding
→ <20,000 platelets/uL: may cause spontaneous
(nontraumatic) bleeding
• Bleeding resulting from thrombocytopenia is associated with
a normal PT and PTT
• Spontaneous bleeding associated with thrombocytopenia most
often involves small vessels.
• Common sites for such hemorrhages:
→ Skin and the mucous membranes of the gastrointestinal and
genitourinary tracts.
→ Most feared is intracranial bleeding
CATEGORIES OF THROMBOCYTOPENIA
DECREASED PLATELET PRODUCTION
• Results from:
→ Conditions that depress marrow output generally
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 ▪ Decreases megakaryocyte number
▪ Eg. aplastic anemia and leukemia
→ Affects megakaryocytes somewhat selectively
▪ Eg. Megaloblastic state, drugs and alcohol
• HIV may infect megakaryocytes and inhibit platelet production
• Myelodysplastic Syndrome
DECREASED PLATELET SURVIVAL
• Can have an immunological or nonimmunologic basis
• Immune Thrombocytopenia:
→ Destruction is caused by the deposition of antibodies or
immune complexes on platelets
→ Normally, antibodies to platelets can recognize self-antigens
(autoantibodies) or non-self antigens (alloantibodies)
→ Alloantibodies can arise when platelets are transfused or
cross the placenta from the fetus into the pregnant mother
• Non-immunologic Thrombocytopenia:
→ Most common causes:
▪ Disseminated intravascular coagulation
▪ Thrombotic microangiopathies
▪ Mechanical injury (prosthetic heart valves)
→ Unbridled, often systemic, platelet activation reduces platelet
life span
SEQUESTRATION
• Normal 30-35% of platelets sequestered by the spleen
• Can rise to 80-90% when the spleen is enlarged
DILUTION
• Due to massive transfusions
• Prolonged blood storage (number of viable platelets decreases)
• Transfusion (plasma volume and red cell mass are reconstituted
but the number of circulating platelets is relatively reduced)
CHRONIC IMMUNE THROMBOCYTOPENIA (ITP)
• Caused by autoantibody mediated destruction of platelets
• Can be primary / idiopathic (absence of any known risk
factors) or secondary (occur in the setting of a variety of
predisposing conditions and exposures
• Secondary Chronic ITP: seen in px with systemic lupus
erythematosus, HIV infection, and B-cell neoplasms such as
chronic lymphocytic leukemia
• Diagnosis of primary chronic ITP is made only after secondary
causes are excluded.
PATHOGENESIS
• The autoantibodies (IgG) SYNTHESIZED IN THE SPLEEN,
most often directed against platelet membrane glycoproteins
IIb-IIIa or Ib-IX
• Antiplatelet antibodies act as opsonins that are recognized by
IgG Fc receptors expressed on phagocytes, leading to
increased platelet destruction
• Splenectomy improves thrombocytopenia because spleen is
the major site of removal of opsonized platelets
MORPHOLOGY
• Principal changes of thrombocytopenic purpura are found in the
spleen, bone marrow, and blood, but they are not specific.
→ Secondary changes related to the bleeding diathesis may be
found in any tissue or structure in the body
• Spleen:
Patho RBC and Bleeding Disorders
→ Normal size,
→ With congestion of the sinusoids and enlargement of the
splenic follicles
▪ often associated with prominent reactive germinal centers
→ Scattered megakaryocytes within the sinuses,
▪ Possibly representing a mild form of extramedullary
hematopoiesis driven by elevated levels of
thrombopoietin.
• Bone Marrow:
→ increased number of megakaryocytes.
→ Megakaryocytes are immature, with large, nonlobulated,
single nuclei (NON SPECIFIC)
• Peripheral blood
→ Abnormally large platelets (megathrombocytes)
CLINICAL MANIFESTATIONS
• Occurs most commonly in adult women younger than 40 years
of age
• Cutaneous bleeding is seen in the form of pinpoint hemorrhages
(petechiae)
→ Prominent in the dependent areas where the capillary
pressure is higher
→ May lead to echhymose
DIAGNOSIS
• History: Easy bruising, nosebleeds, bleeding from the gums,
and hemorrhages into soft tissues from relatively minor trauma
• May manifest first with melena, hematuria, or excessive
menstrual flow
• Lab: Low platelet count, normal or increased megakaryocytes
in the bone marrow, large platelets in the peripheral blood, PT
and PTT normal
MANAGEMENT
• Glucocorticoids (which inhibit phagocyte function), but many
eventually relapse. Given in severe thrombocytopenia
• Splenectomy normalizes the platelet count in about two thirds of
patients, but with the attendant increased risk of bacterial sepsis
• Immunomodulatory agents such as intravenous
• immunoglobulin or anti-CD20 antibody (rituximab) are often
effective in patients who relapse after splenectomy or for whom
splenectomy is contraindicated
ACUTE IMMUNE THROMBOCYTOPENIC PURPURA
• Caused by autoantibodies to platelets
• Disease of childhood (seen in children after viral infection)
• Occurs abruptly – 1 to 2 weeks after a self-limited VIRAL
INFECTION
→ Triggers the development of autoantibodies
• Self-limited – resolves spontaneously within 6 months
→ Without a viral prodrome – persists
▪ Follows a childhood form of adult chronic ITP
DRUG-INDUCED THROMBOCYTOPENIA
• Due to direct effects of drugs on platelets
• Secondary to immunologically mediated platelet destruction
• Quinine, quinidine, vancomycin
→ Bind platelet glycoproteins and create antigenic
determinants that are recognized by antibodies
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• Drugs may also induce true autoantibodies thru unknown Transient neurologic Prominence of acute
mechanisms deficits renal failure
• Thrombocytopenia is also a common consequence of platelet Renal failure
inhibitory drugs
→ Binds to glycoprotein IIb/IIIa → conformational change in GP • Many adult patients with TTP lack one or more of the five
IIb/IIIa → immunogenic epitope criteria
• Some patients with HUS have fever and neurologic dysfunction
HEPARIN-INDUCED THROMBOCYTOPENIA: (so di pa rin ganun ka-established yung differences nila)
• Caused by autoantibodies • In both conditions:
• Occurs in 5% of persons receiving heparin → Intravascular thrombi cause a microangipathic hemolytic
anemia and widespread organ dysfunction
TYPE I THROMBOCYTOPENIA → Attendant consumption of platelets leads to
• Occurs rapidly after the onset of therapy thrombocytopenia
• Resolves despite continuation of therapy • Unlike in DIC, in TTP and HUS:
• Most likely from direct platelet-aggregating effect of heparin → Activation of coagulation cascade is not of primary
importance – lab tests (PT, PTT) are usually normal
TYPE II THROMBOCYTOPENIA
• Occurs 5 to 14 days after therapy begins (or sooner if a person
is sensitive to heparin)
• PARADOXICALLY, leads to life-threatening arterial and
venous thrombosis
• Caused by antibodies that recognize complexes of heparin and
platelet factor 4
→ PF 4 is a normal component of platelet granules
• Antibodies bind to complexes → activates platelets →
thrombosis (even in the setting of thrombocytopenia)
• Unless therapy is discontinued and an alternative nonheparin
anticoagulant is given:
→ clots in large arteries may lead to vascular insufficiency and
limb loss
→ Emboli from DVT can cause fatal pulmonary
thromboembolism

HIV-ASSOCIATED THROMBOCYTOPENIA
Figure 23. Causes and associations of thrombotic microangiopathies
• One of the most common hematologic manifestation of HIV
• Impaired platelet production and increased destruction
THROMBOTIC THROMBOCYTOPENIC PURPURA (TTP)
• CD4 and CXCR4
• Cause: in a plasma enzyme called ADAMTS13 (vWF
→ Receptor and coreceptor of HIV metalloprotease)
→ Found on megakaryocytes → infected → apoptosis → NORMALLY, Degrades very high molecular weight multimers
• HIV also causes B cell hyperplasia and dysregulation → of vWF
predisposes to development of autoantibodies → Absence: multimers accumulate in plasma and promote
→ Autoantibodies opsonize platelets, promoting destruction by platelet activation and aggregation
mononuclear phagocytes in the spleen and elsewhere • Superimposition of endothelial injury -> promote the formation
• In some instances the antibodies are directed against of platelet microaggregates -> exacerbating TTP
glycoprotein IIb-III complexes • Deficiency may be inherited or acquired
• Deposition of immune complexes on platelets may also → Acquired form: autoantibodies inhibit activity of ADAMTS13
contribute to the accelerated loss of platelets
→ Inhertied form: inactivating mutation in ADAMTS13
▪ Onset is often delayed until adolescence and symptoms
THROMBOTIC MICROANGIOPATHIES: TTP AND HUS
are episodic
• Includes thrombotic thrombocytopenic purpura (TTP) and
hemolytic-uremic syndrome (HUS)
• Caused by insults that lead to excessive activation of platelets,
which deposit as thrombi in small BVs
• SAME CLINICAL FEATURES BUT DIFFERENT CAUSES

Table 1. TTP vs. HUS

TTP HUS
Fever Frequent occurrence in
children
Thrombocytopenia Thrombocytopenia
Microangiopathic Microangiopathic Figure 24. Microthrombi present in the heart of a px who died of ttp.
hemolytic anemia hemolytic anemia

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 HEMOLYTIC-UREMIC SYNDROME (HUS)
• Normal levels of ADAMTS13 D. HEMORRHAGIC DIATHESES RELATED TO
• Cause: infectious gastroenteritis caused by E.coli O157:H7 ABNORMALITIES IN CLOTTING FACTORS
– elaborates a Shiga-like toxin • Bleeding due to isolated coagulation factor deficiencies most
→ Toxin is absorbed from inflamed gastrointestinal mucosa into commonly manifests as large posttraumatic ecchymoses or
circulation → alters endothelial cell function → platelet hematomas, or prolonged bleeding
activation and aggregation • UNLIKE THROMBOCYTOPENIA, bleeding often occurs into
• Children and older adults are at highest risk GIT and GUT and into weight-bearing joints (hemarthrosis)
• Hereditary deficiencies typically affect a single clotting factor:
PRESENTATION → Factor VIII – hemophilia A
• Bloody diarrhea → Factor IX – hemophilia B
• With supportive care, complete recovery is possible → vW Factor – von Willebrand disease
→ Irreversible renal damage and death can occur in more • Acquired deficiencies involve multiple factors
severe cases → Factors II, VI, IX, X, protein C – Vitamin K deficiency
→ Multiple factors are consumed and become deficient – DIC
• ATYPICAL HUS → Acquired deficiencies of single factors – caused by
→ Associated with defects in: autoantibodies
▪ complement factor H
▪ membrane cofactor protein (CD46) THE FACTOR VIII-vWF COMPLEX
▪ factor 1 • Most common inherited disorders of bleeding:
→ normally, these three proteins act to prevent excessive → Hemophilia A (factor VIII)
activation of alternative complement pathway → Von Willebran Disease (vWF)
→ deficiencies can be inherited or acquired (d/t autoantibodies)
→ associated with remitting, relapsing course • Factor VIII
• HUS may also be seen following exposures to other agents that → Essential cofactor of Factor IX
damage endothelial cells (e.g. certain drugs and radiation → Converts factor X to factor Xa
therapy)
→ Made in several tissues (sinusoidal endothelial cells and
Kupffer cells in the liver)
C. BLEEDING RELATED TO DEFECTIVE • Circulation → Factor VIII binds to vWF (produced by
PLATELET FUNCTIONS endothelial cells and, to a lesser degree, by megakaryocytes) →
• Inherited disorders can be classified intro three: vWF stabilizes factor VIII (half-life of from 2.4 hours to 12
→ Defects of adhesion hours when bound to vWF in the circulation)
→ Defects of aggregation • Factor VIII interacts with several other proteins involved in
→ Disorders of platelet secretion hemostasis (including collagen, heparin, and possibly platelet
• Among the acquired defects, the clinicially significant are membrane glycoproteins)
caused by: • Most important functions of vWF:
→ Aspirin – potent, irreversible COX-inhibitor; used for coronary → Promotes the adhesion of platelets to the subendothelial
thrombosis matrix.
→ Uremia – defects in adhesion, granule secretion and ▪ This occurs through bridging interactions between platelet
aggregation glycoprotein Ib-IX, vWF, and matrix components
▪ Some vWF is secreted from endothelial cells directly into
BERNARD SOULIER the subendothelial matrix
• Inherited deficiency of platelet membrane glycoprotein → Promotes platelet adhesion if the endothelial lining is
complex Ib-IX (receptor for vwF for platelet adhesion) → disrupted
Defective adhesion of platelets to subendothelial matrix → • Endothelial cells and platelets also release vWF into the
Bleeding circulation.
→ Upon vascular injury, this second pool of vWF binds collagen
GLANZMANN THROMBASTHENIA in the subendothelial
• Autosomal recessive trait → vWF multimers may also promote platelet aggregation by
• Deficiency or dysfunction of GP IIb/IIIa (participates in “bridge binding to activated GpIIb/IIIa integrin
formation” between platelets by binding fibrinogen) • Factor VIII and vWF protein levels are measured by
→Thrombasthenic platelets fail to aggregate in response to
ADP, collagen, epinephrine, or thrombin IMMUNOLOGICAL TECHNIQUES
• Defective platelet aggregation • Mixing the patient’s plasma with formalin-fixed platelets and
• bleeding tendency is often severe ristocetin, a small molecule that binds and “activates” vWF.
• Ristocetin induces multivalent vWF multimers to bind platelet
DISORDERS OF PLATELET SECRETION glycoprotein Ib-IX and form interplatelet “bridges.”
• Characterized by defective release of certain mediators of • The resulting clumping (agglutination) of platelets is measured
platelet activation: thromboxanes and granule-bound ADP in a device called an aggregometer.
• Storage pool disorders

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• The degree to which patient plasma promotes ristocetin-
dependent platelet agglutination reflects the vWF activity of the
sample.
VON WILLEBRAND DISEASE
• Most common inherited bleeding disorder of humans
• Bleeding tendency: mild, often unnoticed
→ until some hemostatic stress (eg. surgery or dental
procedure) reveal its presence
• Most common presenting symptoms:
→ Spontaneous bleeding from mucous membranes (e.g.,
epistaxis)
→ Excessive bleeding from wounds
→ Menorrhagia
• Autosomal dominant disorder; rare autosomal recessive variant
• Three broad categories of vW disease:
→ Type 1 – autosomal dominant disorder; mild to moderate
vWF deficiency
→ Type 3 – autosomal recessive disorder; very low levels of
vWF; severe clinical manifestations
→ Type 2 – autosomal dominant disorder; qualitative defects in
vWF; mild to moderate bleeding
* Type 1 and 3 are associated with quantitative defects
Type 1 von Willebrand Disease
• 70% of all cases
• Incomplete penetrance and variable expressivity
• Associated with a spectrum of mutations
→ Point substitutions → interfere with maturation of vWF
protein or result in rapid clearance from the plasma
Type 3 von Willebrand Disease
• Bleeding characteristics resemble those in hemophilia since
severe deficiency of vWF has a marked effect on the stability of
factor VIII
• Associated with deletions or frameshift mutations involving both
alleles
Type 2 von Willebrand Disease
• 25% of all cases
• With subtypes; Type 2A is the most common
• vWF is expressed in normal amounts but missense mutations
are present → defective multimer assembly
• Missing from plasma are large and intermediate multimers (the
most active forms of vWF)
CLINICAL MANIFESTATIONS
• Defects in platelet function despite a normal platelet count
• Reduced ristocetin cofactor activity**
→ ** Measure of the plasma level of active vWF
• (Remember that vWF stabilizes factor VIII): Deficiency of vWF
→ secondary decrease in factor VIII levels → prolongated PTT
in vWF disease types 1 and 3
→ Adverse complications typical of severe factor VIII deficiency
such as bleeding into the joints are not seen except in rare
type 3 patients
• Wide variability in clinical expression is common
• Due to modifying genes that influence circulating levels of vWF
• Patients with vWF disease facing hemostatic challenges (dental
work, surgery) are treated with:
→ DESMOPRESSIN → stimulates vWF release
Patho RBC and Bleeding Disorders
→ Infustions of plasma concentrates containing factor VIII and
vWF
HEMOPHILIA A (FACTOR VIII DEFICIENCY)
• Most common hereditary disease associated with life-
threatening bleeding
• Caused by mutations in factor VIII*
→ *essential cofactor for factor IX in the coagulation cascade
• Inherited as X-linked recessive trait → affects mainly males
and homozygous females
→ Rarely, in heterozygous females: inactivation of the X
chromosome bearing the normal factor VIII allele by chance
(unfavorable lyonization) → excessive bleeding
• ~30% of px have no family history; disease cause by new
mutations
• Heterogeneity in the causative mutations → Wide range of
clinical severity in hemophilia A
→ <1% normal level – severe disease
→ 2-5% normal level – moderately severe disease
→ 6-50% normal level – mild disease
• As with -thalassemia:
→ Genetic lesions include:
▪ Deletions and nonsense mutations → create stop codons
and errors in mRNA splicing
▪ Inversion involving X chromosome → complete
abolishment of synthesis of factor VIII - most severe
deficiencies
▪ Point mutations in factor VIII → impaired protein function;
may be normal in immunoassay – less common
CLINICAL MANIFESTATIONS
• Symptomatic cases:
→ Tendency toward easy bruising and massive hemorrhage
after trauma or operative procedures
→ “Spontaneous” hemorrhages frequently occur in regions of
the body susceptible to trauma (joints) → hemarthroses
→ Recurrent bleeding into the joints → progressive deformities
→ crippling
▪ Petechiae are characteristically absent
• Prolonged PTT and normal PT
→ An intrinsic coagulation pathway abnormality
• Diagnosis: requires Factor VIII-specific assays
• Precise explanation for the bleeding tendencies of hemophiliacs
remains uncertain
TREATMENT
• Infusions of recombinant factor VIII
→ In 15% of px: develop antibodies (perceived the protein as
foreign) → bind and inhibit factor VIII → can be a very difficult
therapeutic challenge
HEMOPHILIA B (FACTOR IX DEFICIENCY or
CHRISTMAS DISEASE)
• Clinically indistinguishable from factor VIII deficiency
→ Remember that factors VIII and IX function together to
activate factor X
• Due to a wide spectrum of mutations of the gene encoding for
factor IX
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• X-linked recessive trait with variable clinical severity (like
Hemophilia A)
• Prolonged PTT and normal PT
• Diagnosis: assay of the factor levels
• Treatment: infusions of recombinant factor IX
E. DISSEMINATED INTRAVASCULAR
COAGULATION (DIC)
• Excessive activation of coagulation and formation of thrombi in
the microvasculature of the body
→ Coagulopathy can be localized to a specific organ
→ Thrombotic diathesis → consumptions of platelets, fibrin, and
coagulation factors; activation of fibrinolysis
• Acute, subacute, or chronic thrombohemorrhagic disorder
• DIC is not a primary disease but a coagulopathy that
occurs in the course of a variety of clinical conditions
→ A secondary complication of many different disorders
• SSX relating to:
→ tissue hypoxia and infarctions due to myriad microthrombi
→ hemorrhage due to depletion of factors for hemostasis and
activation of fibrinolytic mechanisms; or both
ETIOLOGY AND PATHOGENESIS
Remember the normal process of clotting in vivo:
Figure 25. In vivo, tissue factor is the major initiator of
coagulation, which is amplified by feedback loops involving thrombin (dotted
lines). The red polypeptides are inactive factors, the dark green polypeptides are
active factors, while the light green polypeptides correspond to cofactors.
• DIC could result from pathologic activation of coagulation or
impairment of clot-inhibiting mechanisms
• Two major mechanisms that trigger DIC:
→ release of tissue factor or other, poorly characterized
procoagulants, into the circulation
▪ Sources of procoagulants include placenta in obstetric
complications, tissues injured by trauma or burns, or
mucus from certain adenocarcinomas
→ widespread injury to the endothelial cells
ENDOTHELIAL INJURY
• Endothelial injury can initiate DIC in several ways
• Subtle endothelial injuries can already unleash procoagulant
activity
• Injuries causing endothelial cell necrosis → exposition of
subendothelial matrix → activation of platelets and coagulation
pathway
Patho RBC and Bleeding Disorders
• Widespread endothelial injury may be produced by:
→ Deposition of antige-antibody complexes (SLE)
→ Temperature extremes (heat stroke, burns)
→ Microorganisms (meningococci, ricketssiae)
• One important mediator: TNF
→ Implicated in DIC occurring in sepsis
→ Induces endothelial cells to:
▪ express tissue factor on their cell surfaces
▪ decrease the expression of thrombomodulin
→ up-regulates the expression of adhesion molecules on
endothelial cells → promoting the adhesion of leukocytes →
release ROS and preformed proteases → endothelial cell
damage
• In bacterial infections:
→ endotoxins →stimulate immune cells to make TNF → inhibit
endothelial expression of thrombomodulin and activate factor
XII
→ Antigen-antibody complexes formed → activation of classical
complement pathway → production of complement
fragments → activate both platelets and granulocytes
• In massive trauma, extensive surgery, and severe burns:
→ Major trigger: release of procoagulants (tissue factor)
• In obstetric conditions:
→ Procoagulants are from the placenta, dead retained fetus, or
amniotic fluid → may enter the circulation
• In hypoxia, acidosis, and shock:
→ Cause widespread endothelial injury
• Cancers most frequently associated with DIC:
→ Acute promyelocytice leukemia
→ Adenocarcinomas of the lung, pancreas, colon, and stomach
POSSIBLE CONSEQUENCES OF DIC
• A. Widespread deposition of fibrin within the
microcirculation leading to:
→ ischemia of more affected/vulnerable organs
→ microangiopathic hemolytic anemia
▪ due to red cells squeeze through the narrowed
microvasculature → fragmentation of red cells
• B. Hemorrhagic diathesis
→ Due to consumption of platelets and clotting factors and the
activation of plasminogen
→ Plasmin: cleaves fibrin and digests factors V and VIII →
further ↓ in concentration of clotting factors
→ Fibrinolysis → production of fibrin degradation products →
inhibition of platelet aggregation, fibrin polymerization, and
thrombin
Figure 26. Pathophysiology of DIC.
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 CLINICAL MANIFESTATIONS
• Onset can be:
→ Fulminant (endotoxic shock or amniotic fluid embolism)
→ Insidious and chronic (carcinomatosis or retention of a dead
fetus)
• ~50% are obstetric px with pregnancy complications
→ Reversible with delivery of the fetus
• ~33% have carcinomatosis
• Common patterns worthy of descriptions since it is almost
impossible to detail all the potential clinical presentations:
→ Microangiopathic hemolytic anemia
→ Dyspnea, cyanosis, and respiratory failure Figure 27. DIC; a section of glomerulus which demonstrates several
microthrombi.
→ Sudden or progressive circulatory failure and shock
• Acute DIC (obstetric cases or major trauma)
F. COMPLICATIONS OF TRANSFUSION
→ Dominated by bleeding diathesis
• Most complications are minor and transient
• Chronic DIC (cancer px)
• Most common: Febrile nonhemolytic reaction
• Presents with thrombotic complicationsD
→ Fever and chills, sometimes with mild dyspnea, w/in 6 hours
• DIAGNOSIS:
of a transfusion of red cells or platelets
→ Clinical and laboratory studies
• Symptoms respond to antipyretics and are short-lived
▪ Fibrinogen levels, platelets, PT and PTT, and fibrin
• Reactions are caused by inflammatory mediators from donor
degradation products
leukocytes
• PROGNOSIS:
→ Increases with storage age of the product
→ Depends on the underlying disorder
→ Decreases with measures that limit donor leukocyte
• TREATMENT:
contamination
→ Remove or treat the inciting cause
• MANAGEMENT:
ALLERGIC REACTIONS
→ Meticulous maneuvering between the Scylla of thrombosis
• Severe, potentially fatal allergic rxns in blood products with
and Charybdis of bleeding diathesis
certain antigens are given to previously sensitized recipients
→ Administration of anticoagulatnts or procoagluants in specific
• Most likely to occur in px with IgA deficiency (1:300 – 1:500
settings
people)
→ Triggered by IgG antibodies that recognize IgA in the infused
MORPHOLOGY
blood product
• Thrombi most often found in the brain, heart, lungs, kidneys, ▪ Rare since most px with IgA deficiency do not develop
adrenals, spleen, and liver (in decreasing order and frequency) such antibodies
→ Any tissue can be affected • Urticarial allergic reactions
→ Affected kidneys: small thrombi in glomeruli → reactive → Triggered by an allergen in the blood product recognized by
swelling of endothelial cases in severe cases, microinfarcts, IgE antibodies in the recipient
or bilateral renal cortical necrosis
→ More common (1-3% of transfusions) and generally mild
• “Hyaline membranes” remiscent of ARDS
→ Respond to antihistamines
→ From numerous fibrin thrombi in alveolar capillaries
→ Do not require discontinuation of transfusion
• CNS: fibrin thrombi → microinfacts → simultaneous
hemorrhage → can lead to variable neurologic ssx
HEMOLYTIC REACTIONS
• Endocrine glands:
ACUTE HEMOLYTIC REACTIONS
→ Fibrin thrombi w/in microcirculation of adrenal cortext →
• Caused by prefored IgM antibodies against donor red cells that
probable basis for the massive adrenal hemorrhages in
fix complement
Waterhouse-Friderichsen syndrome
• Error in patient ID or tube labeling that allowsa px to receive an
• Unusual form of DIC in Kasabach-Merrit syndrome (with gian
ABO incompatible unit of blood
hemangioma)
• Pre-existing high affinity “natural” IgM ab → bind to red cells →
→ Thrombi form w/in neoplasm because of stasis and recurrent
rapidly induce complement mediated lysis, intravascular
trauma to fragile blood vessels
hemolysis, and hemoglobinuria → fever shaking chills, and flank
pain
→ Ssx are due to complement mediated lysis > intravascular
hemolysis
→ Osmotic lysis of red cells → hemoglobinuria w/o any of the
other symptoms of a hemolytic rxns
• Severe cases: progress to DIC, shock, acutre renal failure, and
death
• Direct Coombs test – (+)
→ Unless all donor of the donor red cells have lysed

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 DELAYED HEMOLYTIC REACTIONS ▪ Platelet must be stored at room temp → favorable for
• Caused by antibodies that recognize red cell antigens that the bacterial growth
recipient was sensitized to previously (eg., prior blood → Symptoms include:
transfusion) ▪ Fever, chills, hypotension
• Caused by IgG antibodies to foreign protein antigens ▪ Resemble those of hemolytic and non-hemolytic
• Direct Coombs test – (+) transfusion rxns
• Laboratory features: hemolysis (eg., low habptoglobin and → Treatment: start broad-spectrum antibiotics prospectively
elevated LDH) while awaiting lab results
• Severe and potentially fatal rxns in complement activation of • Viral infections (rare)
antibodies to antigens such as Rh, Kell, and Kidd → Occur when the donor is acutely infected
→ Identical to ABO mismatches → Transfusion-related transmission of viruses such as
• Minor ssx in other antibodies that do not fix complement → red ▪ HIV (1 in 2 million)
cell opsonization, extravascular hemolysis, and spherocytosis ▪ Hepatitis C (1 in 1 million)
▪ Hepatitis B (1 in 500,000)
→ Low risk “exotic” infectious agents such as West Nile virus,
TRANSFUSION-RELATED ACUTE LUNG INJURY trypanosomiasis, and babesiosis may also occur
(TRALI)
• Severe, frequently complication REFERENCES
• Factors in a transfused blood product trigger the activation of • Robbin’s 9e
neutrophils in the lung microvasculature • Doc GDM’s ppt
• Incidence: low (1:10,000 transfusions) • Rubins
• May occur more frequently in px with preexisting lung disease
• Pathogenesis: “two hit” hypothesis
→ Priming event → increased sequestration and senitization of
neutrophils → primed neutrophils activated by a factor in
transfused blood product (second hit)
→ Involves endothelial activation
→ Leading candidates for factors implicated as “second hits”
▪ Antibodies in the transfused blood that recognize the ag
expressed on neutrophils
• Most common antibodies associated: those that bind MHC
antigens (particularly MHC Class I)
→ Often found in multiparous women
▪ Generate antibodies in response to foreign MHC ag
expressed by the fetus
• More likely to occr in transfusion of products with high levels of
donor antibodies
→ Fresh frozen plasma and platelets
• Clinical Presentation:
→ Dramatic, sudden onset respiratory failure, during or soon
after a transfusion
→ Diffuse bilateral pulmonary infiltrates seen on chest imaging
– do not respond to diuretics
→ Fever, hypotension, hypoxemia
• Treatment
→ Supportive
• TRALI is impt to recognize
→ Donor products that induce the complication in one px are
much more likely do so in a second
→ Exclusion of multiparous women from plasma donation →
half reduction in the incidence of TRALI

INFECTIOUS COMPLICATIONS
• Any infectious agent can be transmitted but bacterial and viral
infections are most likely to be so
• Bacterial infections:
→ Usually caused by skin flora
→ Contamination occurred at the time that the product is taken
from the donor
→ Significant contaminations/those that produce symptoms:
common in platelet preparations >> red cell preparations

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 Figure 28. Normal B12 metabolism

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