1 HEMATOPOIESIS

Hematopoiesis

Hematopoiesis development of cells of blood system (platelets, WBCs,

RBCs) & supporting structures
Self-renewal constantly replicating & differentiating, blood is constantly
turning over
Development begins early embryogenesis, in first 8 weeks begins
liver/spleen, continues marrow
o Primitive system embryonic yolk sac develops RBCs
o Definitive system development begins in liver/spleen, continues
in late fetus in bone marrow
Axial skeleton primary site for hematopoiesis throughout life
Distal long bones hematopoiesis gradually declines into
adulthood
Dynamic hematopoiesis process adjusted during times of wound healing,
blood loss, infection
Cell Biology

Hematopoietic Stem Cell have properties of self-renewal,

quiescence, multipotency:
o Self-renewal stem cells constantly replicating & turning over
o Quiescence stem cells usually dormant, induced to become active
o Multipotent stem cells can differentiate into 10 different subclasses
(platelets, WBCs, RBCs)
Identification numbers are low, cant be detected through microscopy, ID
relies on cell surface markers, specifically CD34, in addition to Thy-1la and
c-Kit
Origin from aorto-gonadic mesonephric region (AGM) in the ventral aspect
of the dorsal aorta hemangioblast hematopoietic stem cells
Stem Cell Differentiation

Transcription Factors balance of various TFs predisposes stem cells to

differentiate in various ways
Growth Factors from bone marrow environment, interact with cells at
different stages to promote cell growth
o Early development cells stay near cytokine-rich bony spicules,
receive many growth factors
o Late development cells migrate into sinuses, become mature &
ready for use
Stem Cell Lineages

Hematopoietic Stem Cell (HSC) differentiates into

o Common Lymphoid Precursor (CLP) makes B & T lymphocytes,
NK cells
o Common Myeloid Precursor (CMP) makes various myeloid
precursors:
Granulocyte precursor gives rise to neutrophils,
eosinophils, basophils
Monocyte precursor can mature into macrophages

Erythroid/megakaryocyte precursors mature into RBCs,

platelets
BFU/CFU burst forming/colony forming units; more specific CMP stages
before precursors above
Experimental Determination stem cell differentiation studied via mouse
transplantation, cell cultures
Erythropoiesis

Erythropoiesis development of RBCs; process includes:

Hemoglobin major protein component of RBCs,
requiring globins, protoporphyrin, & iron for synthesis
Morphologic Development Stages appearance of RBC in development
related to hemoglobin synthesis
Process: HSCCMPBFUCFU Proerythroblast basophilic polychrom
ortho retic RBC
o Proerythoblast small amount of hemoglobin in RBC
o Basophilic erythroblast more condensed chromatin, more
hemoglobin
o Polychromatophilic erythroblast maximum hemoglobin produced,
nucleus condenses
o Orthochromatophlic erythroblast nucleus fully condensed
o Reticulocyte nucleus of RBC is extruded, some RNA remains
o RBC
Turnover differentiation process takes ~4 days, and lifespan is ~120 days
GM-CSF granulocyte-macrophage colony stimulating factor, cytokine
for BFU/CFU diff
Erythropoietin also plays important role in erythropoiesis, from CFU
onwards
Hemoglobin Formation

, hemoglobin heterodimer present in early embryogenesis (1-2

months)
, fetal hemoglobin present in later fetal development (2-10 months)
, adult hemoglobin (newborn onwards)
Megakaryopoiesis

Megakaryopoiesis generation of platelets small, anuclear cells

Endomitosis nucleus divides, but cell doesnt; leads to single polypoid
nucleus, hi cytoplasmic volume
Platelet formation mature megakaryocyte sheds of platelets without
nuclei
Thrombopoietin (TPO), IL-11 play important role in megakaryocyte
development
Monopoiesis/Granulopoiesis

CFU colony forming unit stem cell common to both

granulocytes/monocytes
GM-CSF important stimulating factor for CMF CFU
Granulopoiesis leads to cells with prominent granules: PMNs, eosinophils,
basophils

Granulocyte Colony Stimulating Factor (G-CSF) primary

cytokine at work
o Basophils & eosinophils have common precursor cell before
undergoing final commitment
o Mast cells similar to basophils, derived from HSC developing in
tissue
Monopoiesis CFU-GM develops to CFU-M monocytes, not too much
morphologic change
o Macrophage Colony Stimulating Factor (M-CSF) primary
cytokine at work
Lymphopoesis

Common Lymphocyte Progenitor (CLP) precursor to lymphocytes

Location maturation takes place outside bone marrow:
o T-lymphocytes takes place in thymus
o B-lymphocytes takes place in bone marrow, then secondary
lymph tissue (nodes, spleen)
Heterogenous although look similar, many different functions, surface
markers, life spans, environs
B-cell development HSCCLPPro/Pre-B mature Bcell activation, plasma cell
T-cell development HSCCLP thymus, differentiate
into , lineages
Interleukins IL-7 important early, later IL-1, IL-2, IL-4

2 RED BLOOD CELLS

Erythropoiesis Overview

Chromatin more condensed as RBC matures: basophilic

erythroblast poly- ortho-chromatophilic
Cell Nucleus becomes extruded to form reticulocyte (has some remaining
RNA), matures to RBC
Reticulocytes not normally seen in peripheral blood, if seen, may indicate
early release of RBCs from marrow
Cytoplasm changes color from purple red as more hemoglobin
produced
Erythopoietin

Erythropoietin (EPO) produced in kidney in response to low hemoglobin &

thus decreased O2 supply
Effect causes erythroid marrow hyperplasia, stimulates RBC production
Increased EPO often a sign of a hemolytic process (sickle-cell, betathalassemia)
Renal failure causes low EPO chronic anemia; normally Hct is negatively
related to EPO, in RF Hct declines b/c of lower EPO

Hemoglobin Types

Embryonic Hgb 22 tetramer, produced in yolk sac

Hemoglobin F has 22 tetramer subunits, is fetal hemoglobin
Hemoglobin A has 22 tetramer subunits, is adult hemoglobin
Hemoglobin A2 has 22 tetramer subunits, a sparser adult hemoglobin
Hemoglobin Physiology

Functions binds O2 in sigmoid fashion (binding cooperativity), binds CO2 to

release O2, binds H+ efficiently at low pH
Affinity d by pH (dumps O2 in acidic environment), 2,3 DPG (high
altitude), and temperature (when something drops, O2 affinity increases)
Fetal Hemoglobin binds less 2,3 DPG and thus has higher O2 affinity
Conformations exists in T tense state and R relaxed state
o T Tense State nonaccepting, low O2 affinity (deoxygenated)
o R Relaxed State accepting, high O2 affinity (partially
oxygenated) binding cooperativity
Hemoglobin Variants

High Affinity favor R state, delivers less O2 to tissues, decreased affinity

for 2,3 DPG, increased erythrocytosis, higher Hgb
Low Affinity favor T state, delivers more O2 to tissues; see cyanosis
clinically, slate-gray color skin c/w cyanosis
Unstable increased susceptibility to oxidative stress, heme/globin
complex is unstable Heinz bodies
o Heinz bodies intracellular precipitates in RBCs that have
weakened heme/globin binding
o Hemolysis occurs when Heinz bodies bind to RBC
membrane damages RBC, phagocytosis
M Hemoglobins abnormal heme environment favoring T state right
shifted curve, pseudocyanosis (brown-slate skin, no respiratory distress even
though right shifted)
Heme Biosynthesis

RBC

Location in RBC hemoglobin, but also in liver heme

enzymes (cytochrome P-450s, catalase)
Process several steps:
1) Glycine + Succinyl CoA makes aminolevulinate acid (ALA) in
mitochondria; *rate-limiting* under negative feedback by heme
2) ALA leaves mitochrondria, enters cytosol, eventually converted
to coproporphyringen III
3) Coproporphyringen III re-enters mitochondria, Fe incorporated into
porphyrin ring by ferrochelatase
Sideroblastic Anemia X-linked lacks ALA synthase cant make ALA
during heme biosynthesis iron deposits around nucleus of erythroblasts
(ringed sideroblasts)
Porphyrias patient lacks
other heme biosynth enzymes overproduction of heme precursors incr
eased excretion, phosensitivity, ab pain, neuro complications
Cytoskeleton
Spectrin & Ankryin important proteins giving RBC biconcave shape

Hereditary spherocytosis central pallor lost, phosphoplipid bilayer leaky,

cell swells due to Band 3 or Ankyrin mutation
Hereditary elliptocytosis spectrin abnormal, RBC becomes elliptically
shaped
RBC Metabolic Pathways

Na-K ATPase sodium-potassium pump, inhibited by ouabain

Glycolysis generates 2 ATP, also NADH/NADPH, moth balls and fava beans
can alter metabolism resulting in hemolysis
Pentose-Phosphate shunt uses G6P to make
NADPH synthesize glutathione (used by catalase)
RBC Degradation

Lifespan normally ~120 days, abnormal is <90 days

o Short Lifespan caused by spherocytosis, sickle
cells, thalassemic RBCs
o Dx can see a compensation with increased reticulocytes
Senescent/Abnormal RBCs degraded by RES reticulo-endothelial system
(spleen 1o)
Degradation hemoglobin binds to haptoglobin, complex is then
transported to liver:
o Globin Degradation globin broken down into AAs
o Heme Degradation broken down into Fe + biliverdin, which is
reduced to bilirubin
o Fe Degradation picked up by ferritin, can accumulate over time to
make hemosiderin, iron overload results in hemosiderin deposits in
other organs
Hemoglobin Hemolysis Hgb unbound to haptoglobin is oxidized
to methemoglobin
o Met-heme binds to albumin or hemopexin enters liver
RES, heme catabolism
o Hemopexin measure of hemolysis (like haptoglobin), low levels =
high hemolysis
Bilirubin Degradation

Conjugated Bilirubin esters added to bilirubin by bilirubin-UDPglucuronyl transferase (BUGT)

o Water soluble conjugated form very water soluble, can then
be excreted
o Gilbert Syndrome, Crigler-Najjar Syndrome BUGT mutated,
hyperbilirubinemia
Unconjugated Bilirubin binds to albumin, less water soluble, elevated in
jaundice
o Jaundice from increased bilirubin load on immature liver, Tx by UV
light

3 ANEMIA

Anemia

Anemia a decrease in RBC/hemoglobin mass, resulting in deficient O2carrying capacity of blood

Symptom anemia is considered a symptom of an underlying disease rather
than a 1o disease itself
Evaluation includes Hx, CBC, peripheral blood smear:
o Clinical/Hx SOB, fatigue, angina, hypotension; cardiac compensation
(inc HR, vasoconstrict)
o CBC complete blood count, summarizing RBCs, WBCs, platelets
o Peripheral blood smear examine morphology microscopically
CBC Variables

Quantitiative include RBC number, hemoglobin content, & hematocrit:

o RBC Number [RBCs] in blood; normally 4.0-5.0 *1012/liter
o Hemoglobin Content [hemoglobin] in blood; normally 13-15 g/dL
o Hematocrit % of blood occupied by RBCs; normally 40-45%
Qualitative includes properties of RBCs in blood:
o Mean Corpuscular Volume (MCV) ratio of Hct/RBC count gives
vol. of single RBC; 90 f
o Mean Corpuscular Hemoglobin (MCH) ratio of Hgb/RBC
count gives Hgb vol. per RBC
o Mean Corpuscular Hemoglobin Conc. (MCHC) ratio
of Hgb/Hct gives [Hgb] per RBC
o RBC Distribution Width (RDW) variance in size of RBCs
Anemia Classification: Reticulocytes

Reticulocytosis if bone marrow is responding to anemia, will

see reticulocytes
o Reticulocyte slightly larger than mature RBC, with some nuclear
material remaining
o Polychromasia immature RBCs wont be as red as normal; some
variation in color (purplish)
o Reticulocyte Index amount of reticulocytes made during anemia,
assess bone marrow response
Low Index marrow
not fxn properly; hypoproliferative or maturation
abnormality
High Index marrow functioning; cause is rather from blood
loss, hemolysis, hematinic
Anemia Classification: RBC Size

RBC Size size of RBC will give clues to anemia cause:

o Macrocytic anemia large RBC > 100f
Megaloblastic anemia impaired DNA synth; due
to folate/B12 deficiency, or Rx SE
Non-megaloblastic anemia unrelated to DNA
synth; bleeding/hemolysis, liver dz
o Microcytic anemia small RBC <80f
Decreased Hemoglobin defects in globin chain
production, heme synthesis
Inherited thalassemias (defective hemoglobin)

Acquired iron deficiency, chronic disease, lead poisoning,

medication SE
o Normocytic anemia normal RBC 80-100f
Reticulocytes > 2% - acute hemorrhage, hemolysis
Acute Hemorrhage GI/menstrual bleeding commonly
w/ Fe deficiency as end result
Hemolytic anemia decreased RBC longevity
(membrane Hgb defect)
Reticulocytes < 2% - bone marrow failure, kidney disease
(low EPO)
1o leukemia, lymphoma, myeloma, myelodysplastic
syndromes
2o renal disease, chronic illness, medication SE,
metastatic tumor affect EPO
Anemia of Chronic Disease

Underlying inflammatory state infection, rheumatoid disorders,

malignancies
Hepcidin prevents iron absorption in duodenum and release from
macrophages
Other cytokines lead to impaired proliferation of progenitor cells and
blunted response to EPO
Labs low serum iron, normal/elevated serum ferritin, decrease EPO
Anemia & Location of Destruction

Intravascular Hemolysis destruction within transport system of blood;

RBCs usually break apart schistocytes
o Mechanical destruction heart valve, disrupted blood fow shearing
RBCs
o Vascular Damage DIC, TTP, HUS
o Immune-mediated severe antibody reactions, PNH
o RBC defects sickle cell disease, PNH (paroxysmal nocturnal
hemoglobinuria)
Extravascular destruction outside of transport system, caused by intrinsic
property of blood spherocytosis w/ elevated RDW:
o Hemolytic anemia IgG bound to RBC
o Heinz body anemia damaged RBCs from heme/globin precipitate
o Hereditary spherocytosis abnormal RBC membrane
Labs
o Extravascular polychromatophilia, increase retic, erythroid
hyperplasia, increased bilirubin, absent haptoglobin, nlincreasedHbg, variably increased LDH
o Intravascular polychromatophilia, increase retic, erythroid
hyperplasia, increased bilirubin, reduced haptoglobin,
increasedHbg, increased LDH

4
5 NUTRITIONAL ANEMIAS

Nutritional Anemia

Nutritional Anemia anemia resulting from lack of essential substrate

normally ingested
Necessary nutrients include iron, folate, vitamin B12/B6,
niacin, Vit A/C/E, copper, AAs, cobalt
Iron Physiology

Iron

Anemia Prevalence most common type of nutritional anemia (think

anemia Tx = iron supplements)
Distribution normal content is ~3-4gm, 70% in heme, 30% stored, <0.2%
in plasma:
o Hemes (70%) hemoglobin & myoglobin carry most of bodys iron
o Ferritin/hemosiderin (30%) most of remaining non-heme, storage
forms of iron
o Transferrin (<0.2%) iron in plasma, bound to transferrin protein
Metabolism absorbed in GI tract; used and re-used repetitively; 30% used
in liver:
o Non-heme proteins liver makes cytochromes
o Tissue heme proteins liver makes myoglobin
Absorption US diet 10-15mg Fe/day, only about 1-2 mg absorbed;
although more absorbed if needed
o Heme iron absorbed intact
o Non-heme iron gastric acid reduction of Fe3+ to Fe2+, presence of
absorption inhibitors (grain, tea, egg yolks) or enhancers (VitC) must
consider diet in causes of anemia
Hepcidin regulator of iron homeostasis, limits GI absorption/recycling
Transport carried in plasma by transferrin protein (can carry many Fe
ions)
o Total iron binding capacity
300 ug Fe/dl; increased during deficiency, pregnancy, estrogen
o Decreased capacity during infammation, tumor, liver disease,
nephrotic syndrome
o Transferrin saturation proportion of available iron-binding sites
occupied by Fe atoms (serum FE/TIBC)*100%
o Cell Import transferrin binds to transferrin receptor; whole
compound endocytosed, Fe dumped
Storage mainly stored in ferritin (less stable, more soluble, small capacity)
and hemosiderin (opposite)
Excretion no physiologic mechanism; just lost when cells lost
(bleeding, GI/renal epithelium slough)
Deficiency Anemia
Sx can be asymptomatic early, or can have fatigue, weakness, DOE,
pallor, light-headed
Sx of underlying cause GI problems, bleeding, psoriasis
Exam Findings glossitis (tongue swollen), angular cheilosis (cracked
corners of mouth),
esophageal webs (dysphagia), koilonchyia (fingernails fatten),
blue sclera,
gastric atrophy, pica (craving for ice chewing)

Dx conduct CBC (Hgb, Hct, MCV, RDW), measure serum iron/ferritin,

transferrin saturation
o RDW increased during Fe deficiency; take on weird shapes
o MCV slowly decline as less Hgb made
o Serum ferritin low level is Dx, but normal level doesnt rule out
Iron Stores 1st lose storage forms (ferritin/hemosiderin), next
in transport forms (transferrin), last RBC
Bone Marrow Aspirate gold standard, Dx is absence of intracellular iron
(no Prussian blue staining)
Etiology can be from increased iron requirements (physiologic/pathologic),
or low supply
o Physiologic stresses growth, pregnancy, lactation (lost in breast
milk)
o Pathologic stresses blood loss
o Inadequate supply low Fe in diet, impaired absorption, abnormal
transferrin
Treatment treat underlying cause, give oral iron replacement (ferrous
sulfate), or IV iron dextran
Megaloblastic Anemia

Megaloblastic Anemia anemia caused by a defect in DNA

synthesis larger RBCs
Common Causes lack of vitamin B12 or folic acid
Peripheral Blood Smear looks the same for vitamin B12 (cobalamin) and
folic acid deficiencies:
o RBCs anemia, increased MCV (anisocytosis), increased
RDW, poikilocytosis (variation in shape)
o WBCs PMNs hypersegmented, mild-to-moderate leukopenia
o Platelets mild-to-moderate thrombocytopenia
Bone Marrow Aspirate hematopoietic cell hyperplasia (all 3 cell lines)
DDx congenital dyserythropoetic anemia, erythroleukemia, Rx SE
(contraceptive), macrocytosis (liver dz)
Clinical Manifestations Sx of anemia (above), and effects of impaired DNA
synthesis:
o Epithelial tissues glossitis (swollen, smooth tongue),
angular cheilosis (cracked corner mouth)
o Neural tissues vitamin B12 deficiency only periph. neuropathy,
dorsal columns/cord degeneration, optic atrophy, psychiatric disorders
Megaloblastic Anemia: Vitamin B12 Deficiency

Function Vitamin B12 is essential cofactor for 2 enzymatic reactions:

o Methyltransferase convert homocysteine methionine;
form tetrahydrofolate DNA synth
o Adenosylcoblamain Mutase converts methylmalonylCoA succinyl CoA
Source produced only by vitamin B12-producing microbes (bacteria, fungi);
humans get from diet
o Intrinsic factor protein in stomach conjugating vitamin B12, to
absorb in GI tract
o Intestinal bacteria make vitamin B12 too distally for absorption

Content average US diet 5-7 ug vitamin B12/day, 2-5 mg total body content
(1 mg stored liver)
Mechanisms include inadequate diet or inadequate absorption:
o Inadequate diet if strict vegetarian, or breast-fed infants of mothers
w/ B12 deficiency
o Inadequate absorption lack of gastric acid, intrinsic factor (pern.
anemia), reduced receptors, pancreatic insufficiency, Zollinger-Ellison
syndrome, nonfunctional TCII, NO inactivation of B12
Dx obtain serum B12 level, also elevated
homocysteine/methylmalonic acid (uncatalyzed reactants)
Schilling Test used to localize site of metabolic defect causing B12
deficiency:
o Initial patient given oral radiolabeled vitamin B12 and injection of
normal B12
o Stage I record amount of radiolabeled B12 excreted in 24 hour urine
collection (normal = 92%)
o Stage II (if Stage I abnormal) patient given oral (intrinsic
factor/pancreatic enzyme/etc) + radiolabeled B12 if normal, problem
is with a lack of intrinsic factor/pancreatic enzyme/etc
o Stage III 7-10 days of Abx, if due to bacterial overgrowth, dose will
be excreted in a 24 hour urine collection
o Pancreatic insufficiency patient is coadministered pancreatic
extract w/ radiolabeled B12
Intrinsic Factor / Parietal Cell Antibodies occasionally the problem
Treatment replenish B12 (IV/oral), may need pancreatic extract, exogenous
intrinsic factor
Folic Acid dont give, can exacerbate neuropsychiatric manifestations of
B12 deficiency
Megaloblastic Anemia: Folate Deficiency

Folate used in coenzyme tetrahydrofolate methylated

when homocysMet; used for dUMPdTMP
Content 5-10 mg in body, most stored in liver; children/pregnant require
more in diet
Source obtained in diet green leafy vegetables, yeast, legumes, fruits
Absorption in small intestine, no specific transport protein; binds
nonspecifically
Enterohepatic recirculation re-uses/redistributes folate
Intracellular remains w/ cell throughout cells lifespan
Mechanism through inadequate intake, increased requirements,
malabsorption, drugs, congenital
o Inadequate intake low folate levels in diet
o Increased requirement in children, pregnancy, lactation, hemolysis
o Intestinal malabsorption sprue, Crohns disease
o Drugs ethanol, barbiturates, sulfa drugs
Dx obtain serum folate level; more reliably RBC folate level,
also homocysteine/methylmalonyl CoA
o Homocysteine should be elevated in folic acid deficiency (reaction
not catalyzed)
o Methylmalonic acid should be normal in folic acid deficiency (not
involved in this process)

Tx treat underlying problem, give folate supplements; prophylactic

folate in pregnant women

6 CONGENITAL HEMOLYTIC ANEMIAS

Erythropoiesis

Turnover RBCs survive 100-120 days, macrophages remove 1% each day

Hemolysis premature/increased RBC destruction; Hgb released (high
serum conc), binds to haptoglobin
Heme breakdown into Fe + unconjugated bilirubin
Compensation can have increased RBC production during increased
hemolysis resulting in normal Hbg
Hemolytic Anemia

Hemolytic Anemia state of hemolysis in which increased RBC production is

outpaced by destruction
Severity highly variable, may not manifest until adulthood, can be lifelong
hemolysis or just risk of it
Family History very important in establishing diagnosis
Congenital/Acquired two main categories of hemolytic anemia:
o Congenital sickle cell, thalassemia, spherecytosis, G6PD deficiency
o Acquired autoimmune hemolysis, DIC, TTP, drug-induced
Vascular Scope can be intravascular or extravascular:
o Intravascular RBC destruction within the circulation
o Extravascular caused by ineffective erythropoiesis in bone
marrow, or spleen/RES overdrive
Causes hemolytic anemia caused by intrinsic/extrinsic factors:
o Intrinsic RBC wall structure, cytoplasmic enzyme defects,
abnormal Hgb
o Extrinsic oxidative effects (RBC wall damage), immune-mediate
(RBC wall damage),
mechanical destruction shear forces of turbulent fow

Hemolysis Lab Evaluation

RBC Destruction shows as high LDH, plasma Hgb, & unconjugated

bilirubin, also low haptoglobin
RBC Production has a high reticulocyte count
Specific Labs blood smear, Coombs test
(autoimmune), Hgb electrophoresis, RBC fragility & enzyme
levels, specific DNA mutation studies

Congenital Hemolytic Anemia: Thalassemia

Thalassemia reduced/absent synthesis of or globin chains

Consequences imbalanced globin chains defective Hgb synthesis RBC
damage
Distribution mainly mediterranean, along equatorial region
Pathophysiology reduced synthesis of globin production imbalanced
globin chain synthesis and reduced normal Hb production excess of free or
unpaired globin chains damage to RBC precursors hemolysis
Classification include and types:
o Thalassemia - >175 point mutation types, affects
any/all two copies of one beta gene, Ch11
Demographic Mediterranean & African
Onset after birth, when made for Hgb A
Affects adult Hgb A1 (since 22), but not Hgb A2 (22)
or Hgb F (22)
No tetramers excess cannot form
tetramers precipitates in RBCs hemolysis
Thalassemia Minor mutation in one
gene asymptomatic
Thalassemia Intermediate mutation in both genes,
leading to less production
Thalassemia Major loss-of-function mutation in both
genes, no production
Electrophoresis shows decreased Hgb-A1, increased A2
and F
o Thalassemia many point mutations, affects
any/all four copies of two alpha genes, Ch16
Demographic Asian & African
Tetramers excess , can form , tetramers soluble 4,
4 less damage
Onset before birth, since need in fetus too
-/ silent carrier, normal production
-/-, --/ Thal- trait, mild decrease in chain
production, mild anemia
--/- Hgb H disease 4 = Hgb H, formed in excess,
severe hemolytic anemia
--/-- Hydrops fetalis fatal to fetus, Hgb Barts = 4;
non-functional
Diagnosis family Hx, microcytosis, blood smear,
hemolysis, Hgb electrophoresis, DNA studies
o Microcytosis +/- anemia hallmark of Thalassemia
o Blood smear can see microcytosis, hypochromia, target cells,
Heinz bodies
o RBC Destruction evidence see above
o Hemoglobin electrophoresis assess levels of Hgb types
Treatment monthly transfusions, iron chelation (iron overload often
fatal)
o Folic Acid supplementation since excess erythropoiesis uses folate
o Splenectomy to prevent hyperactive RES
o Bone marrow transplant for severe refractory
Congenital Hemolytic Anemia: Sickle Cell

Sickle Cell Disease caused by single mutation in

gene Val replaces Glu at 6th AA
Sickle Trait have only one of two genes mutated, heterozygous S (HbSA)
Sickle Disease can be Hb-SS, Hb-SC, Hb-SThal:
o Hb-SS homozygous sickle cell Hgb
o Hb-SC heterozygous sickle cell Hgb + heterozygous Hgb C
o Hb-SThal heterozygous S + heterozygous Thal (thus only 1 viable
gene, Hgb S)
Physiology sickle cell Hgb can have
unwanted polymerization form sickle cells, can occlude vessels
Prevalence 1 in 10 African Americans carry trait, 1/400 have SS Dx =
80,000 US patients
Clinical Exam asymptomatic until Hgb F declines (6 months), variable
severity, infections
Sickle Disease Crises various acute syndromes of sickle cell disease:
o Pain crisis from vaso-occlusion in bones, splenic infarcts, strokes
Aggravators from infection, acidosis, hypoxia, dehydration
o Visceral sequestration crisis under age 2, acute hypoxia RBCs
pool in liver/spleen/lungs
Respiratory tract infection - aggravates
Respiratory failure from atelectasis, creates life-threatening
emergency
Tx blood exchange
Splenic sequestration in young pts rapidly enlarging
spleen, ab pain, worsening anemia recurs, tx w/ splenectomy
o Aplastic anemia crisis from parvovirus infection rapidly
enlarging spleen; Tx splenectomy
o Hyperhemolytic crisis hemolytic symptoms acutely
Sickle Cell Disease Infections repeated splenic infarcts may effectively
cause splenectomy by age 2
o Bacteria streptococcus, H. infuenza, meningococcus susceptible
o Prophylaxis need to give prophylactic penicillin, specific
immunizations
Treatment give RBC transfusions, folate,
also hydroxyurea (increases HbF, reduce severity)
o Refractory experimental bone marrow transplant, exchange
transfusions for crises
Congenital Hemolytic Anemia: Spherocytosis & Elliptocytosis

Hereditary Spherocytosis (HS) RBC membrane defect

of vertical interactions
o Deficiencies in spectrin, ankyrin, protein 3, protein 4.2
o Binding defect primarily defect in binding of spectrinProtein
4.2
o Prevalence more common, 1/5000
Hereditary Elliptocytosis (HE) - RBC membrane defect
of horizontal interactions
o Deficiencies in Protein 4.1, Protein 3, Glycoproteins C&D
o Binding defect primarily defect in binding of spectrinankryin
o Prevalence more rare, 1/25,000; usually African & Mediterranean

HS/HE Pathophysiology RBC becomes more spherical, less

deformable occludes spleen vasculature
HS/HE Clinical Exam commonly ASx, mild/mod anemia, jaundice, hemolysis
recurrence, splenomegaly
HS/HE Diagnosis blood smear (spherocytes/elliptocytes), osmotically
fragile RBCs, increased MCHC
HS/HE Tx often none, can give folate, and splenectomy can be curative
(then vaccines, prophylaxis)
Congenital Hemolytic Anemia: G6PD Deficiency

Glucose-6-Phosphate Dehydrogenase enzyme needed to maintain high

level of reduced glutathione
o Location gene located on X chromosome
o Demographic several variants in African American/Mediterranean/SE
Asian males
Glutathione used to reduce peroxides H2O + oxidized glutathione;
prevents damage
Oxidative Damage RBC damage in oxidizing conditions (drugs) leads to
hemolysis
Causes infection, fava beans, and drug SEs
Diagnosis acute intermittent hemolysis, Hgb normal between crises,
measure G6PD/oxidized glutathione
Treatment stop drug! Consider splenectomy
Congenital Hemolytic Anemia: Pyruvate Kinase Deficiency

Pyruvate Kinase glycolysis enzyme generating ATP; lack causes rigid

RBC, hemolysis
Diagnosis chronic ongoing hemolysis, splenomegaly, neonatal jaundice
Treatment - splenectomy

7 PERIPHERAL BLOOD SMEAR

Blood Smear Technique

Smear Concentration smear blood across slide horizontally, so density is

from high low
Examination examine where not too packed, but not so sparse to create
morphological distortion artifact
WBC Differential zig-zag through good field until 100 WBCs counted; put
into categories
o Left Shift means more premature WBCs in blood, due to active
infection/appendicitis
RBC Morphology includes
anisocytosis, poikilocytosis, polychromatophilia, hypochromia:
o Anisocytosis variation in size (1+ just detectable, 4+
macro/microcytes together)

Poikilocytosis variation in shape (1+ occasional abnormal cell, 4+

50% cells abnormal)
o Polychromatophilia retained RNA (1+ 1-2 blue cells per oil field, 4+
50% cells blue) retics
o Hypochromia increased central pallor (1+ just detectable, 4+ only
faint RBC rim left)
Platelet Estimation 9-20 per oil field = 200,000-400,000/uL = normal
Normal Morphology

RBC should have pale orange-pink appearance with central pallor 1/3-1/2
diameter of cell
o Too dense RBC overlap, cells become thicker & dont uniformly have
central pallor
o Too sparse RBCs lack central pallor, too fattened out
WBC PMNs most common; myelocytes usually indicate infection, any blast
form = definite infection
Neutrophil Maturation Series

Myeloblast precursor to granulocytes/monocytes:

o Large ovoid eccentric nucleus, scant cytoplasm
o Several clear nucleoli unlike a lymphocyte (no nucleoli)
o Reddish-purple granules in scant cytoplasm
Promyelocyte myeloblast differentiates into this, precursor to
granulocytes:
o Larger cell size more abundant cytosplasm than myeloblast; about
2x RBC diameter
o Reddish-purple granules indicate myeloid lineage
o Several clear nucleoli unlike lymphocytes
Myelocyte promyelocyte differentiates into this, precursor to granulocytes:
o More condensed nucleus only occupies 50% of cell size;
eccentric
o Blue azure granules in cytoplasm, appears smoother
Metamyelocyte myelocyte differentiates into this, precursor to
granulocytes:
o Indented oval nucleus resembles kidney bean, 50% of cell size
o Absent nucleoli chromatin is now very clumped
o Pink cytoplasm neutrophilic granules completely color cytoplasm
Myeloid Band form precursor to granulocytes:
o C-shaped nucleus eventually will begin to lobulate, but no
indentations yet
o Pink cytoplasm characteristic of granulocyte
Granulocytes final stage of neutrophil maturation
o Polylobular nucleus with many identations, 2-5 lobes
Vs. lymphocytes lymphocytes have much darker nucleus, no nucleoli; most
closely resemble myeloblast
Vs. basophilic normoblast (RBC precursor) like promyelocyte, but no
granules & smaller overall size
Granulocytes

Monocyte larger cell size, lighter horseshoe/kidney nucleus, cytoplasm

usually light w/ some vacuoles

Eosinophil has bright red cytoplasm due to acidophilic granules; 2-3

nuclear lobes
Basophil has dark cytoplasm due to basophilic granules (5HT, histamine,leukotrienes), 2-3 nuc. lobes
Neutrophil pink to purple granules in cytoplasm; most common; 2-5
nuclear lobes, Barr body nuc. notch
Lymphocytes

Small lymphocyte look like myeloblast, but no nucleoli, darker nucleus

Large lymphocyte look like a promyelocyte, but no nucleoli; very scant
granules in cytoplasm
Atypical lymphocyte weird elongated dense nuclei; often seen in viral
infection (mononucleosis)
Neutrophil Abnormalities

Hypersegmentation more than 5 lobes in PMN is

abnormal, Dx of megaloblastic anemia (B12, folate)
Leukemoid Reaction WBC response to infection, >30,000; also can
be leukemia
o Vacuolization cytoplasmic vacuoles seen in PMNs
o Left-shift PMN precursors (myelocytes, etc) can be seen in blood
Pelger Huet Anomaly bi-lobed nucleus; benign inherited disorder; can
mimic leukemia/myelodysplasia
Dohle Body aggregates of RER seen in PMN cytoplasm during infection;
looks like a bluish platelet
Platelets

Platelet small faint doo-hickies; red/purple granules

Abnormalities can be too large (macrothrombocyte) or no granules
(agranular), also gray:
Gray platelet syndrome congenital disorder; decreased granules, larger
than normal size
RBC Abnormalities

Leukoerytrhoblastic RBCs see RBC precursors (erythroblasts w/

nuclei); hemolytic anemia
o Howell Jolly bodies dark granules similar to basophilic stippling;
can occur in left-shift here
Macrocytes very large RBCs
Elliptocytosis RBCs elliptically shaped
Spherocytosis RBCs dont have central pallor; spherical shape
Basophilic Stippling pathologic precipitation of ribosomes in RBC; dark
granules
o Disordered heme synthesis such as in lead intoxication,
thalassemia, etc can cause
Schistocytes fragmented RBCs, look like bites taken out of them; often
mechanical shearing defects
Hemoglobin C Disease show target cells and box car cells with
condensed hemoglobin (pill shape)
Sickle Cell crescent-shaped RBCs
Tear Drop Cells RBCs look like tear drops, Dx thalssemia

Target Cells excess of RBC membrane causes bulging in the center of

central pallor target sign
o Thalassemia Dx by target cells
Rouleaux Formation stacking of RBCs due to protein
coat hypergammaglobulinemia
RBC Agglutination RBC antibodies bind, RBCs cluster and meld together

8 COAGULATION
Normal Circulation

Vascular space blood sequestered in fuid phase balanced

by procoagulant & anticoagulant components
Platelets in inactive form, resemble disc-shaped structures, no tissue factor
(TF)
Tissue Factor contained in monocytes, fibroblasts, and injured
endothelium, not platelets
Coagulation: Initiation
1)

Injury: endothelial lining disrupted, blood exposed to subendothelial cells

and TF-bearing fibroblasts
2) Platelet adhesion: von Willebrand Factor (VWF) tethers platelet to site of
injury, connecting
glycoprotein 1b/FIX receptor of platelet
to collagen of subendothelium
3)
4)

Tissue factor complex: Exposed TF binds to plasma FVII, activates to FVIIa

FX Activation: TF/FVIIa complex activates FX FXa (free FXa in blood
deactivated by antithrombin)
5) FV Activation: TF/FVIIa/FXa complex activates FV FVa, FVa/FXa complex
forms prothrombinase
FIX Activation: TF/FVIIa also activates FIX FIXa, which moves onto
platelet surface
(FIXa isnt rapidly degraded by antithrombin the way FXa, FVa/FXa are)
6)

Thrombin Activation FVa/FXa complex

activates prothrobin (FII) thrombin (FIIa)

Deactivation Tissue Factor Prothrombin Inhibitor (TFPI) can bind to complexes

in 3 & 4, deactivate
Coagulation: Amplification
1)

Platelet Activation Thrombin (FIIa) activates platelets through protease

activated receptors (PARS)
2) FV expression & activation activated platelets express more FV on
surface, thrombin activates to FVa

FVIII activation thrombin also activates FVIII FVIIIa on non-PARS sites on

platelets
FXI activation thrombin also activates FXI FXIa on platelet surface
Fibrin formation thrombin also activates circulating fibrinogen fibrin,
but doesnt stop bleeding yet
Coagulation: Propagation (QUIZ: Absent in Hemophilia!)
3)

1)

Tenase formation activated FIXa (initiation #5) binds to activated FVIIIa on

platelet surface
2) FX activation tenase activates FX FXa on platelet surface (compare
initiation #4, endo surface)
3) Prothrombinase formation FVa & FXa complex on platelet surface
(compare initiation #6, endo)
4) Thrombin burst prothrombinase activates
thrombin (FII FIIa); thrombin burst
Coagulation: Clot Formation
1)

Fibrin clot formation thrombin burst activates

circulating fibrinogen fibrin, and now forms clot
Recombinant FVII

rFVIIa a recombinant FVII synthesized; bypasses need for FVIII & FIX for
clotting
o QUIZ: Direct activation of FV & FX leads to thrombin burst
directly
Formation several steps:
o FVII on chromosome 13, synthesized in liver, once activated
becomes FVIIa, same as rFVIIa
o Plasmid vector FVII plasmid isolated, introduced to baby hamster
kidney (BHK) cells
o rFVIIa formation forms in BHK cells, secreted into media BHK cells
growing on
Usage high dose rFVIIa can induce hemostasis (clotting) in hemophiliacs
TF independent rFVIIa doesnt work on non-activated platelets, only
activated ones near injury
TF dependent rFVIIa can also complex with TF, activates FX FXa,
complexes FVa thrombin

9 ACQUIRED HEMOLYTIC ANEMIAS

General Principles
Hemolytic anemias result from shortened RBC survival due to increased rate of destruction
Pathogenesis anemia occurs when rate of destruction exceeds ability of marrow to replace
RBCs
o Bone marrow can increase rate of production 6-8 times baseline rate
o Absence of anemia doesnt rule out hemolytic disorder b/c bone marrow can
compensate
Classification by Extrinsic Factor
Immunohemolytic Anemia autoimmune response attacks RBCs:
o Maltransfusion blood transfusion incompatible

o
o

Hemolytic disease of newborn

Warm & Cold-reactive autoimmune hemolytic anemias warm/cold agglutinins
Traumatic Anemia mechanical trauma on RBCs causes hemolysis
o Prosthetic valves/cardiac abnormalities create turbulent flow, shear RBCs
o Hemolytic uremic syndrome RBCs sheared in glomeruli
o Thrombotic thrombocytopenic Pupura (TTP)
o Disseminated intravascular coagulation
Other causes hemolytic anemias can arise from infectious agents,
chemicals/drugs/toxins, physical agents, hypophosphatemia, paroxysmal nocturnal
hemoglobinuria, spur cell anemia, Vitamin E deficiency
Classification by Site of Hemolysis
Intravascular RBCs lysed in vessels:
o Dx verified by hemoglobinemia, hemoglobinuria, hemosiderinuria
o Sx include constitutional symptoms, tachycardia, back ache, Sx related to renal failure
o Haptoglobin decreased
Extravascular RBCs lysed outside of vessels (often spleen):
o Sx jaundice, splenomegaly (RBCs lysed in spleen)
o Haptoglobin typically normal, or slightly decreased
Hemolysis Lab Evaluation
Basics look at Hgb, Hct, also calculate MCV (normal to elevated) and conduct reticulocyte
count
Peripheral Blood Smear look for polychromatophilia, microspherocytes, fragmented RBCs, etc
Bone marrow aspirate assess for erythroid hyperplasia
Serum unconjugated bilirubin assess heme breakdown products, for jaundice
Serum lactate dehydrogenase (LDH) elevated in damaged tissues
Plasma haptoglobin binds hemoglobin if it is free in plasma, decreased haptoglobin when
bound
Urine hemosiderin occurs after 7d of hemolysis when renal tubular epithelial cells can absorb
no more
Cold agglutinin test detects cold agglutinating antibodies (IgM) in patients serum with normal
RBCs
Direct Antiglobulin Test (DAT)/Coombs Test detects presence of IgG or C3 bound to RBC
o Autoimmune hemolytic anemia hallmark is the positive Coombs test
o Process wash patients RBCs free of plasma, add antiglobulin reagent, centrifuge, look
for agglutination
Indirect Antiglobulin Test/Indirect Coombs detects autoimmune hemolytic anemia as well
o Process incubate patients serum with normal blood, look for reaction (x-fusion
compatibility)
Immunohemolytic Anemias
Autoimmune hemolytic anemia (AIHA) antibody/complement binds to RBC membrane
antigens
1o/2o disorders can occur 1o idiopathic, or 2o to underlying disease/drug
Anti-RBC Antibodies divided into 3 categories:
o IgG warm antibodies bind to RBCs at 37C, but fail to agglutinate cells
o Cold agglutinins almost always IgM, clump RBCs at cold temperatures
o IgG Donath-Landsteiner antibodies bind RBCs in cold, then complement cascade if
warm
Self/Non Igs on RBCs can be direct against self or non-self antigens, or neo-antigens (unusual
non-self)
Warm-Antibody AIHA

Prevalence about 70% of AIHAs

Physiology when IgG binds RBC membrane thru spleen & engulfed by macrophages
o Spherocyte results when part of cell membrane removed by macrophage in spleen
o Result spherocytes eventually cleared by extravascular mechs in spleen
Etiology can be 1o idiopathic, or 2o to lymphoproliferative dz, CT dz (SLE), immune
deficiency, Rx
o Immune deficiencies including AIDS, and common variable immunodeficiency
o Drugs classically alpha-methyldopa
Sx can be asymptomatic or anemic Sx, also jaundice, splenomegaly
Clinical Exam increased RR, jaundice, pallor, splenomegaly, underlying autoimmune
disorder Sx
o Autoimmune disorder Sx fever, lymphadenopathy, skin rash, HTN, renal
failure, petchiae
Tx treat underlying cause first, and then varying levels of care:
o No therapy if patient has well-compensated hemolytic processes
o Folic Acid give for all patients, to ensure RBC production
o Steroids mainstay Tx, thought to interfere with Fc receptor of Igs
o RBC transfusion only for severe refractory cases, risk of hemolytic reaction
o Splenectomy also for refractory, when corticosteroids fail
o IVIg may increase RBC survival by saturating Fc receptors on macrophages, cant deal
w/ RBC
o Immunosuppressive therapy including danazol, vinca alkaloids, rituximab
Cold Agglutinin AIHA
Physiology usually IgM antibodies against RBCs, binding at cold temp, targets I/i blood group
antigen
Hemolysis after binding to RBCs, IgM activates complement cascade C3b binds,
phagocytosis by
hepatic macrophages (rather than splenic RES cells)
Severity dependent on Ig titer & ability to initiate complement activation
Chronic/Acute Disorder two main forms of disease:
o Chronic Disorder common, occurs in 5th decade or later, often concurrent with Blymphocyte neoplasm
o Acute Disorder rare, occurs younger, often complication
of infectious dz (mycoplasma anti-I, mono anti-i)
Sx cold-induced acrocyanosis (fingertips, toes, nose, ear lobes), anemia Sx, coldassoc. hemoglobinuria
Clinical Exam signs of anemia, jaundice, splenomegaly
Labs low Hgb, hemolysis signs (reticulocytes, spherocytes, bilirubin, polychromatophilia,
LDH), blood smear, DAT, agglutinins
o Blood smear will show RBC agglutination, spherocytes, polychromasia
o DAT positive for C3 only (not IgG)
o Cold agglutinins test positive, obviously
Tx treat underlying cause first, and then varying levels of care:
o Avoidance of cold exposure most basic conservative Tx, usually pretty effective
o Combination chemotherapy for refractory, can be helpful
o Glucocorticoids rarely helpful
o Splenectomy rarely indicated
Paroxysmal Cold Hemoglobinuria (PCH)
Demographic rare AIHA disorder, occurs in children following viral
illness, tertiary/congenital syphillis

Physiology caused by IgG binding RBCs at cold temperatures, but falls off at 37oC
Sx paroxysmal symptoms following cold exposure: fever, leg/back/abd pain, rigor,
hemoglobinuria, renal failure
Dx incubate normal RBCs w/ patients serum vs. normal serum, measure lysis at 4oC, 37oC
o DAT usually negative, since IgGs elute off cells in normal temp
o P blood group antigen Donath-Landsteiner antibody Dx of PCH
Tx self-limited, give supportive care, avoid cold temperatures
Drug Induced Immune Hemolytic Anemia
Hapten Mechanism drugs binds to RBC membrane, IgG against drug binds RBC destroyed
o DAT tests positive for IgG
o Drugs most commonly penicillins; also ampicillin, methicillin, cephalosporins
Immune Complex Mechanism drug binds to plasma protein/Ig, forms immune complex, binds
RBC
complement activation & hemolysis
o Prevalence most common drug-induced immune hemolytic anemia, usually IgM
antibody
o DAT positive for complement (no IgG IgM here)
o Drugs most commonly quinine, phenacetin, antihistamines, insulin, acetaminophen,
sulfa
Autoantibody Mechanism offending drug induces autoantibody formation, usually for Rh
antigen
o DAT positive for IgG
o QUIZ: Drugs most commonly alpha-methyldopa, also ibuprofen
In Vivo Sensitization Mechanism drug binds RBC membrane antigen, forms immune complex,
anti-drug Ig then binds to neo-antigen drug on RBC hemolysis
o DAT positive for IgG
Non-Immune Hemolytic Anemias
Fragmentation Hemolysis (Microangiopathy) result of mechanical shearing of RBCs from
damaged microvasculature, cardiac abnormalities, AV shunts, turbulent flow, drugs (cyclosporine,
cocaine)
o Diagnosis peripheral blood smear shows fragmented RBCs
o Treatment treat underlying condition, supportive care
Hypersplenism functionally hyperactive spleen too much sequestration of all blood cells
o Splenomegaly not synonymous; splenomegaly caused by wide variety of factors
o DDx vascular congestion, infxn, inflammatory dz, hemolytic anemia, neoplasm, storage
disorder, amyloidosis, sarcoidosis, structural abnormalities (cysts)
Infection can be several mechanisms of hemolysis (direct attack, hypersplenism induction,
immune, toxin release, altered RBC surface)
Paroxysmal Nocturnal Hemoglobinuria
PNH acquired clonal disorder of hematopoietic stem cells producing defective RBCs
o Defective RBCs have an unusual susceptibility to complement-mediated hemolysis
o Pig-a gene mutation affects glycosylphophatidylinositol (GPI) linkage in RBC
membranes
Clinical Exam increase w/ age (2nd decade onwards), can have hemolytic anemia Sx,
pancytopenia, Fe def, venous thromb.
Assoc. Diseases concurrent w/ aplastic anemia, myelodysplastic syndrome,
acute myeloblastic leukemia
Labs evidence of anemia in CBC, normal morphology, variable bone marrow, also:
o Leukocyte alkaline phosphatase (LAP) test is low, (only occurs with leukemia or
this)

Sucrose & Hams acid hemolysis RBCs more susceptible to lysis under these stress
tests
o Flow cytometry can analyze for PIG-linked antigens
Tx try to correct anemia, prevent thrombosis, stimulate hematopoiesis, give eculizumab:
o Correct anemia steroids, iron supprlement, folate
o Prevent thrombosis anticoagulants, thrombolytics
o Stimulate bone marrow hematopoiesis transplant, antithymocyte globulin
o Eculizumab monoclonal antibody against C5 interferes w/ hemolysis & thrombosis
Other Causes of Non-Immune Hemolytic Anemias: liver disease, severe burns, copper deficiency, druginduced oxidative damage

10 HEMOSTASIS
Hemostatic System

Hemostasis blood maintained in fuid phase thru balance

of procoagulant & anticoagulant components
Hemostatic Plug injury needs to be rapidly recognized and occluded,
and clots needs to be amenable to remodeling during healing to
allow normal blood flow in recovery (prevent ischemia distal to injury)
Hemostatic System has cellular components, adhesion molecules,
pro/anticoagulants, fibrinolytic:
o Cellular components include platelets and endothelial cells,
monocytes/macrophages:
Platelets hemodynamic barrier at injury site, locus for
coagulation reactions, SM contraction (ATP, 5-HT; stop bleed)
Endothelial cells contribute to normal anticoagulant state of
vessel
Antithrombin III bound to cell surface, prevents
coagulation
Tissue plasminogen activator released into blood,
breaking up clots
Thrombomodulin activates protein C, anticoagulation
NO, prostacyclins (PGI2) also inhibit coagulation
o Adhesion molecules such
as von Willebrand Factor, fibronectin; allow platelets to attach
o Procoagulants signal clotting cascade, including zymogens (V) &
co-factors (Va) leading to fibrin clot
o Natural Anticoagulants reduce the amount of thrombin produced
or directly inhibit thrombin action
o Fibrinolytic system factors controlling clot remodeling
Vascular Injury

Von Willebrand Factor giant multimeric protein binding

to subendothelium during injury, stored in platelet granules and in plasma

GpIb Receptor VWF binds

to Glycoprotein GpIb receptor in subendothelium, uncoils & exposes
multiple binding sites for platelet adhesion

Platelet receptors have a GpIb receptor binding VWF, also have

a GpIIbIIIa receptor for aggregation
Platelet Activation binding to subendothelial causes partial activation and
once exposed to tissue factor (TF) on surface of endothelial cells, fblasts,
monocytes
Thrombin generated by expsorure to TF; activates platelets by binding
to
PAR1/PAR4 protease activated receptors thrombin binds & activates
platelets morphologic change
Activated platelets undergo morphological change, release alpha &
dense granules into local milieu
Tenase & prothrombinase help activate thrombin release from
platelet = thrombin burst
Recruitment attract more platelets to forming plug
Platelet Aggregation, Amplification, Clot

GpIIbIIIa on platelet, binds fibrin, which then

binds GpIIbIIIa of circulating platelets aggregation
Extrinsic System involve TF and Factor
VII (think subendothelium initiation) initiates
Intrinsic System involve Factors XI, IX, VIII (think platelet
activation) amplifies, doesnt initiate
Contact System uses other cofactors, involved more in modulating
inflammatory response
Clotting Cofactors several factors:
o Factor VIII forms tenase, activates Factor X prothrombinase
o Factor V aids prothrombinase in activating Factor II
(prothrobin) thrombin
o Thrombin Feedback thrombin catalyzes XI XIa of intrinsic
pathway amplification
Fibrin Assembly

Activation thrombin
clips fpA & fpB from fibrinogen forms protofibrils
Fibrin Formation protofibrils can complex vertically & horizontally to
form fibrin
Covalent crosslinks form between protofibrils, make fibrin strong
& resistant to plasmin breakdown
o Factor XIII catalyzes formation of crosslinks between protofibrils
Anticoagulation

Thrombomodulin on endothelial surface; binds thrombin & inhibits

thru antithrombin III
o Antithrombin III binds thrombomodulin-thrombin complex; inhibits
Factor Xa no clot
Protein C activated by a thrombinthrombomodulin complex PCa inactivates Factor Va & VIIIa

Protein S can bind to Protein C to act as a cofactor; binding to C4b BP

inactivates Protein S
Fibrinolysis

u/t-PA tissue plasminogen activator, activates plasminogen plasmin,

breaks down fibrin into D-dimers
o
D-dimers indicates the

presence of a complete clot formation, good lab test to indicate that

clotting has occurred and plasmin has acted to degrade fibrin crosslinks
TPA inhibitor stops TPAs action on plasminogen, keeps clot
Alpha-2-antiplasmin/TAFI inhibits action of plasmin, keeps clot

11 BLEEDING DISORDERS
Bleeding Exam

Severity/Timline is patient having mild bleeding, or profuse? How long has

this been a problem?
Location bleeding can be mucocutaneous (nosebleeds), joints/muscles,
or surgical/trauma sites
o Hemophilias/Clotting Factor Deficiency bleeding
into muscles/joints (hemarthrosis)
o Platelet, VWF, Vessel Disorders
usually mucocutaneous bleeding (nose, gums, periods)

Single Organ/System could be a neoplasm, ulcer, vascular

abnormality
Gingival bleeding in addition to platelet disorders, can be vitamin C
deficiency or gingivitis
Bruising Exam

Ecchymoses a bruise noted on exam; should note:

o Site knees/elbows are common sites for
bruising, back/abdomen are uncommon sites
o Spontaneous vs. Traumatic key defining characteristic
o Severity how large? Is it palpable? How frequently do they occur?
o Coloring different bruise colors mean different Dx:
Purple/blue most likely a true physiologic ecchymosis
Red could be Cushing syndrome, excess steroids., senile
purpura, UV exposure
Jet black warfarin-induced skin necrosis
Rashes include petechiae & purpuras:
o Petechiae common sign of thrombocytopenia
o Purpura larger/thicker, a sign of vasculitis
Bleeding Sites

Fibrinolytic Tissues certain tissues have high fibrinolysis (urinary tract,

endometrium, mucosal, GI)
Hemoptysis/Hematemesis/Melena rarely the presenting sign of patient
w/ 1o bleeding disorder
Menorrhagia heavy or prolonged menstrual bleeding; often from
1o bleeding dz; need medical Tx
Obstetric bleeding if transfusions, iron Tx, or hysterectomy necessary postpartum, could be 1o bleed dz
Bleeding Patterns

Spontaneous/Provoked pay attention to bleeding during tooth extraction,

surgery, trauma, childbirth
Age of first bleed earlier onset = inherited hemostatic disorder; later
= inherited bleeding disorder
Bleeding Patterns variety of bleeding disorders related to different
bleeding patterns
o Mucocutaneous disorder of platelets, VWF
o Joint/Muscle related to severe hemophilias or clotting factor
deficiencies
o Injury-related usually a mild-moderate hemophilia, or
mild clotting factor deficiency
o Umbilical Stump usually disorder of fibrinogen
o Impaired Wound Healing usually Factor XIII deficiency
Bleeding Timing acute = platelet/VWF defects, delayed = coagulation
factor def (thrombin)
Bleeding History

Hospital Visits for bleeding ask patient if this ever was necessary
Significant Bleeding Sx ask if transfusions were necessary, Hx or
anemia/iron deficiency

Medication Hx anticoagulants (warfarin,

heparin), OTCs (aspirin), herbs (gingko, St. Johns wort)
Nutritional Hx are there deficiencies of vitamin
K? vitamin C? general malnutrition?
Family Hx spontaneous bleeding, excessive surgical bleeding, anemia/Fe
deficiency, inheritance
Common Acquired Disorders liver disease, renal failure, vitamin K
deficiency, leukemia
Physical/Lab Exam

Oral Mucosa look for wet purpura, petechiae

Skin look for ecchymoses, petechiae, telangiectasias, bleeding around
venipuncture sites
MSK look for joint deformities, limited mobility
Labs include CBC, Ferritin, and many platelet function studies:
o CBC Hbg, Hct, MCV, platelet count, smear (morphology, bone
marrow dz)
o Ferritin look for Fe deficiency anemias
o Platelet function tests in addition to blood smear & platelet count,
many tests (below):
Platelet Function Tests

Bleeding time normal less than 9 minutes;

measures 1o hemostasis (VWF, platelet)
Platelet aggregation assess response to thrombin, ADP, epinephrine,
collagen
o GpIIaIIIb defect platelets wont clot in response to anything really
Prothrombin Time (PT) isolate plasma w/ some platelets,
add thromboplastin (TF source) clot time
o Pathways Measured measures process of extrinsic system
(VII) thrombin fibrin
o Not measured nothing to do with contact factors (XI, XII)
Activated Partial Thromboplastin Time (aPTT) isolate plasma w/
platelets, phospholipid, activator and calcium added
o Pathways Measured measures contact factors & intrinsic pathway
o Not measured initial steps of extrinsic pathways
Thrombin Clotting Time (TCT) isolate plasma w/ platelets, thrombin
added
o Use tests whether fibrinogen is intact & fibrin clot being formed
o Pathways Measured fibrinogen fibrin clot
o Not Measured anything upstream of thrombin in clotting cascade
o Heparin, FDPs can artificially prolong clotting time
o Warfarin does not affect clotting time
D-dimers & Fibrin Degradation Products assess for presence of fibrinlysed clot = d-dimer & FDP
o Use positive test indicates that clotting system has proceeded to
complete & is fully intact
o Pathway Measured fibrinolysis, furthest downstream = cross-linked
clot FDP & d-dimers
o DVT/Pulmonary Embolus can be detected through this test, as
excess FDP in blood

Lab Value Interpretations Unexpected Values, Factor XIII, Empiric

Unexpected Value first and foremost, repeat test many can be screwed
up
Factor XIII Deficiency patient will present w/ obvious bleeding disorder,
but normal tests (prob w/ heal)
Empiric Treatment consider if patient obviously bleeding badly

despite negative labs

12 CONGENITAL COAGULATION FACTOR

DEFICIENCIES
Hemophilia A Factor 8 deficiency, sex-linked
Hemophilia B Factor 9 deficiency, sex-linked
Hemophilia C Factor 11 deficiency
SQ vs. IM Injections SQ injections okay in hemophilia, IM injections are not!
Hemophilia A

Hemophilia A a Factor VIII deficiency, sex-linked disorder, gene mutation

o Prevalence 1/5000 male births, 30% are de novo mutation, 70%
family Hx
o Sex-Linked on X chromosome sons of hemophilic M are okay,
daughters obligate carriers
sons of hemophilic F 50% hemophilic,
daughters 50% carriers

Clinical Exam excessive bleeding after a circumcision, deep cut, dental

extraction; muscle/joint bleeding
Lab Dx usually has prolonged aPTT test (VIII = intrinsic),
but normal PT (extrinsic unaffected) & TCT
o Factor VIII Activity after obtaining prolonged aPTT, pinpoint w/
specific assay (not 9, 11, 12)
o Factor VIII Deficiency cutoff below 50% of normal range
Male Severity range from asymptomatic severe:
o ASx, VIII > 20% - asymptomatic usually, until a major surgery or
trauma
o Mild, VIII 5-10% - unlikely to spontaneously hemorrhage, but risk
during trauma/surgery
o Moderate, VIII 1-5% - common soft-tissue bleeding
o Severe, VIII <1% - spontaneous muscle/joint hemorrhages
Female Severity F carriers have 50% Factor VIII usually,
but Lyonization can bring down to low as 25%
Hemarthrosis

Acute Hemarthrosis hemorrhaging into joint space, common in severe

hemophilia
o Warning signs earliest Sx tingling sensation & warmth in joint
space; severe pain hours later

Cartilage destruction blood proteases penetrate synovial space,

cause destruction
Chronic Hemophilic Arthropathy vicious cycle of rebleeding, leading to
joint space destruction leading to synovial thickening and adhesion,
hemosiderin deposits causing infammation, fibrovascular proliferation,
subchondral cyst formation, bone collapse andankylosis
Invasive procedure precautions IV infusion of factor if doing ABGs,
LP, bronch; immunizations dont require infusion but should be given subQ
Hemophilia B

Hemophilia B a Factor IX deficiency, sex-linked disorder, gene mutation

o Prevalence 1/30,000 male births, 30% are de novo mutation, 70%
family Hx
o Sex-linked same deal as Hemophilia A above
Clinical Exam hard to Dx before 6 mo, due to naturally low factor IX
(Vit K depdendent) in neonates
Lab Dx same as Hemophlia A, but for Factor IX
Severity same as Hemophilia A
Hemophilia C

Hemophilia C a Factor XI deficiency, not-sex-linked, gene mutation

o Prevalence most common in Ashkenazi Jews
o Not sex-linked inherited in autosomal recessive fashion
Clinical Exam more variable, milder Sx usually after major
surgery/trauma; no spontaneous joint/muscle bleeds
Lab Dx same as Hemophlia A, but for Factor XI
Factor VII Deficiency

Prevalence rare autosomal recessive disorder; from liver disease or

vitamin K deficiency
Clinical Exam similar to Hemophilia A, but milder
Lab Dx has a normal aPTT & TCT but a prolonged PT
Common Pathway Factor Deficiencies

Common Pathway Factors include Factor II (thrombin), Factor V &

X (prothrombinase)
Prevalence all are rare autosomal recessive disorders
Clinical Exam involve soft tissue bleeding and hemarthroses
Lab Dx has a prolonged PT and aPTT, but normal TCT
Congenital Fibrinogen Defects

Hereditary Afibrinogenemia no clottable (functional fibrin) or no antigeni

c (fibrin protein)
Hereditary Hypofibrinogenemia low clottable (functional fibrin)
and low antigenic (fibrin protein)
Hereditary Dysfibrinogenemia low clottable (fxnl fibrin),
but normal antigen (protein); cirrhosis
Clinical Exam has hemophilia presentation but less hemarthroses
o Clottable fibrin when falls below 50 mg/dL, bleeding more likely to
manifest

Thrombotic events some dysfibrogenemias do paradoxically

present with thrombotic events
Lab Dx has prolonged everything, since final step in clotting pathway
Factor XIII Deficiency

Factor XIII used in creating covalent crosslinks between fibrins in clot;

deficiency = premature lysis
Prevalence rare autosomal recessive disorder
Clinical Exam umbilical stump bleeding, intra-cranial hemorrhage, and
soft tissue bleeds
Lab Dx has normal everything, since clot forms fine, just lysed too early
o Urea clot solubility test shows rapid clot dissolution due to lack of
crosslinks
o Threshold clot solubility test only detects factor XIII levels <4-10%,
this is okay, since only 410% needed for normal life

Von Willebrands Disease

Von Willebrand Factor used for anchoring platelets to site of vascular

injury; deficiency = VWD
o Subunits has many subunits
Factor VIII binding region allows for transport of Factor VIII
in blood
Gp1b receptor binding region binds to platelet
receptors & subendothelium
o Thrombotic Thrombocytopenic Purpura (TTP) VWF plays
important role here
o Factor VIII Carrier serves as intravascular carrier protein for factor
VIII to prevent cleavage
Prevalence most common congenital bleeding disorder
Clinical Exam shows mucocutaneous bleeding (epistaxis, mucosal bleed,
menorrhagia, dental bleed), postpartum hemorrhage, rare
spontaneous hemarthrosis (only in type 3 when factor 8 level < 5%)
Lab Dx no one definitive test, instead assess bleeding time, Factor VIII
activity, VWF antigen/activity
o Factor VIII activity measured same as Hemophilia A
o VWF antigen assay just an ELISA test for VWF antigen
o Ristocetin Cofactor Assay measures VWF activity, atbx exposes
Gp1b receptor on VWF from patient plasma agglutinates normal
platelets (not from patient) and compared to standard curvers
o Ristocetin-Induced Platelet Aggregation Assay similar test,
except w/ patients own platelets, and low level
of ristocetin assesses if Gp1b receptor is abnormally sensitive (will
agglutinate)
Assesses for Type 2B VWF deficiency!
Von Willebrands Disease Types

Type I vWD most common, all multimers present but quantity reduced
o Factor VIII Activity, Ristocetin cofactor activity, vWF antigen
all reduced

Type II vWD qualitative defect in vWF, loss of specific multimers; several

types:
o Type 2A most common, mutation in A domain, impaired assembly
& secretion of vWF; large and intermediate multimersreduced
o Type 2B assessed by enhanced RIPA (above), spontaneous platelet
binding thrombocytopenia; caused by point mutation in A1
domain to result in increased and spontaneous binding
to GpIb; mulitmer analysis shows loss of highest molecular
weightmultimers
o Type 2N mimics hemophilia A, autosomal recessive, almost
normal vWF but cant protect Factor VIII reduced; mutation in
factor VIII binding domain
o Type 2M has decreased vWF binding to platelets, due to
unprocessed vWF, decreased activity and normal multimer distribution
Type III almost all vWF & Factor VII depleted, autosomal
recessive, Sx include both platelet type immediate bleeding and hemophilic
type delayed bleeding
Treatment for Coagulation Factor Deficiencies

Fresh Frozen Plasma can partially replace missing coagulant in

emergency, get up to about 15%, cant be routinely used because of large
volume required
Cryoprecipitate contains Factors 8, 13, vWF Tx these deficiencies; not
used in Western world anymore because specific factors now readily available
DDAVP synthetic vasopressin, induces Factor VIII & vWF release from
storage sites Tx these defs but doesnt work for IIb and III
Factor Concentrate for Factors 8, 9, 13, vWF

13 INTRO TO RADIATION ONCOLOGY

Radiation Oncology the use of radiation in the treatment of cancer
Radiation Basics

EM Spectrum radio (low freq) (micro, light, x) therapy rays

(high freq) cosmic (very high freq)
Photons include gamma rays, x-rays; deep penetration, (hi energy = hi
penetration)
Electrons interact directly with tissues; skin dose high (Tx skin cancer, or
tissue close to surface)
Protons & Carbon Nuclei deepest penetration, can kill healthy &
hypoxic cells; but $$$$
o Deep tumors treated with photons, since these penetrate deep
without causing skin damage
Teletherapy

Cobalt Machine antiquated, but uses radioactive cobalt to exposure patient

to radiation (not used anymore)
Linear Accelerator electrons accelerated & made very energetic, direct @
metal target to make x-rays
Brachytherapy

Brachytherapy treatment of cancer via placement of radioactive

sources next to tumor
Inverse square rule intensity is the inverse square of the distance (2x
distance, 4x loss of intensity)
Pros allow for much more radiation given to tumor compared to normal
tissue
Cons making for a uniform dose is difficult
Permanent/Temporary radioactive sources can be permanent or
temporary (minutes to days)
Methods plastic tubes with beads, seeds, high dose rate with probe
Radiation-Cell Interaction

Direct electrons can damage DNA & cell membrane directly (30% of
damage)
Indirect electrons can create free radicals (hydroxyl, superoxide,
peroxide) which then damage cell (70% of damage)
o Radiation duration free radicals exist
for microseconds milliseconds after radiation
o Biological effects occur over hours/days/years after radioactive
exposure
Cellular Responses to Radiation include a number of outcomes:
o Irreversible block cell never changes/grows = senescence
o Apoptosis programmed cell death, 1-2 days after; less common
o Mitotic Death tumor cells attempt to undergo mitosis, and death at
this stage
o Colony formation cells which survive can go on to form colonies
about 3 days after exposure
Radiation Effects on DNA

Single Strand Breaks well-repaired by cell, since template of other strand

exists
Double Strand Breaks less well-repaired, no template to work off
Single dose curve acute bolus of radiation more efective at killing
cells more double strand breaks
Repeated fraction curve chronic low radiation doses less
efective DNA repair machinery works on
single strand breaks to fix before other breaks occur

Fluorodeoxyuridine inhibits sublethal damage repair and allows for

repeated fraction dosing
Cell Death Mechanisms

Mitotic Cell Death damaged cells okay until they try to divide, then
die may take a while to do this
Apoptosis DNA damage activates cell death pathway; lymphocytes &
spermatocytes sensitive
o Lymphomas & seminomas can be destroyed via apoptosis after
radiation Tx
Cell Membrane Signals cell membrane has many life/death signals, which
radiation Tx can target
Radiation Effects in Cells

Genetics some cells inherently more sensitive to radiation than others

Oxygen status cells with more oxygen will have more damaging free
radicals after radiation hypoxic cells are resistant to radiation
Cell Cycle cells in M phase more sensitive to radiation than cells in S
phase
Chemical Modifiers certain drugs in cells can act as
radiation protector/sensitizer
Radiation Physics effects of radiation also dependent on physics:
o Kind of radiation carbon nuclei & protons much more damaging
than electrons, photons
o Speed of dose acute boluses much more damaging than slow
fractions
o Amount and total time of dosing
o Area of radiation obviously determines extent of damage
Radiation Effects at Tumor/Organ Level

4 Rs of Radiation Biology Repopulation, reoxygenation, redistribution,

repair
o Repopulation tumor cells can grow back during a course of
radiation
o Reoxygenation tumor O2 increases as cells die (less
competition) more & more effective
o Redistribution cell cycle distribution changes become more
aligned
o Repair cells can repair damage between fractions
Hyperfractionation if normal cells repair damage better than tumor cells,
can fractionate radiation,
counting on a speedy normal cell recovery but a lagging
tumor cell recovery
Accelerated Fractionation more frequent doses of same radiation
amount; thus more total radiation;
reduce the treatment time in order to keep overall
treatment radiation the same and prevent tumor growth
during treatment

Chemical Modifiers include radiation sensitizers & protectors:

o Sensitizers for tumor cells, hypoxic cell sensitizers,
chemotherapeutic agents, molecularly targeted therapies
o Radiation protectors for normal cells, scavenge free radicals,
prevent cytokine damage (anti-infammatory)
Normal Tissues

Parallel organs damage to small fraction has no toxicity like in lung and
liver
Serial organ damage to small fraction produces toxicity like esophagus and
spinal cord
Therapeutic Index want to maximize this in order to give effective
radiation therapy:
o Single-dose radiation small therapeutic index (about 1.2)
o Fractionated radiation much larger therapeutic index (e.g. 1.2^30
= 36)

o Chemical Modifiers also aim at raising therapeutic index

Radiation Fractionation

Single Large Dose used when small part of organ can afford to be
damaged; similar to ablation
Repeated Fractionated Doses used when organ cannot afford to be
damaged, need normal cells to heal
Radiation Failure

Tumor Size cant give enough radiation to kill every tumor stem cell without
intolerable damage
Tumor physiology hypoxic tumor cells resistant to radiation; tumor cells
may be better at recovering
Radiation Success

Not due to initial damage, only due to prolonged recovery of tumor cells
Normal cells migrate back into irradiated field
Cancer cells not as good at repairing DNA damage
Tumor is more dependent on new vasculature which may be more sensitive to
radiation

14
15 ACQUIRED BLEEDING DISORDERS
Coagulation Initiation Extrinsic Pathway
1) Tissue Factor exposed on damaged endothelium, and on activated macrophages at site of
injury
2) Factor VII binds to TF, complexes & becomes activated to Factor VIIa
3) Factor X binds to Factor VIIa/TF complex, becomes activated to Factor Xa
4) Factor II (prothrombin) proteolytically cleaved by Factor Xa Factor IIa (thrombin)
5) Thrombin helps lead to clotting, but extrinsic pathway isnt enough needs intrinsic feedback
Coagulation Amplification (Feedback) Intrinsic Pathway
1) Thrombin converts Factor XI XIa
2) Factor XIa converts Factor IX IXa
3) Factor IXa feedback onto Factor X Xa, helps reinforce clotting cascade
Coagulation Propagation (Cofactor Catalysis)

Tenase tenase requires Factor VIIIa, but once activated will catalyze Factor X Xa

Prothrombinase requires Factor Va, and will catalyze Factor II

(prothrombin) IIa (thrombin)

Thrombin Feedback thrombin will feedback on Factor VIII (tenase) and Factor
V ( prothromb)
Contact System Pre-Intrinsic Pathway

Contact System occurs before intrinsic pathway, but any defect doesnt result in clinical
bleeding
1) Factor XII PK coverts to Factor XIIa
2) Factor XIIa activates Factor XI intrinsic pathway
Coagulation Pathway Inhibitors

Tissue Factor Pathway Inhibitor (TFPI) inhibits action of Factor VIIa (TFbinding), stops extrinsic

Antithrombin inhibits action of Factor Xa & Thrombin stops common pathway using
thrombin

Weak interaction physiologic antithrombin has a very weak inhibition, fails to totally
stop path

Heparin changes conformation of antithombin makes for

a strong antithombin action

Protein C combines with cofactor Protein S, proteolytically degrades Factor VIII & V,
stops intrinsic

Protein S cofactor which needs to be in free form (not bound to C4b) to work

o Bound Protein S unable to complex with protein C, and intrinsic pathway continues
Clot Formation Fibrin Assembly
1) Thrombin acts to cleave fpA & fpB off fibrinogen forms insoluble fibrin
monomer (protofibril)
2) Fibrin monomers assemble into a fibrin polymer (clot)
3) Factor XIIIa acts on fibrin polymer to crosslink monomers clot stronger
Clot Degradation Plasmin Assembly
1) Plasminogen activated to plasmin
2) Plasmin proteolytically cleaves fibrin polymer clot lysis and D-dimer release
3) D-dimer released via plasmin, can be assayed to verify entirely intact clotting system, embolus
presence
Coagulation Pathway Assays

aPTT uses the contact system to initiate intrinsic clotting pathway in test tube

PT uses extrinsic clotting pathway

Acquired Bleeding Disorders
DIC

Disseminated Intravascular Coagulation (DIC) simultaneous thrombin & plasmin activity

Process underlying disorder activates coagulation systemic coagulation:

Widespread fibrin deposition from systemic coagulation, leads to subacute

thrombosis

Consumption of platelets & clotting factors more commonly leads to acute

hemorrhage

Blood Smear will show schistocytes (from RBCs shearing through fibrin) and platelet absence

Etiology can be acute DIC and subacute DIC:

Acute DIC triggered by infection (sepsis), obstetric complications, trauma

Subacute DIC usually a malignancy, obstetric (retained dead fetus), vascular

thrombotic dz

Clinical Exam present with paradoxical acute hemorrhage and subacute thrombosis

Dx CBC shows lowered platelets, prolonged PT/aPTT; confirmed by elevated D-dimer/FDPs

Tx must treat underlying condition, then worry about restoring normal blood status:

Tx underlying condition antibiotics, surgery, chemotherapy, embolization; dzspecific Tx

APML give all trans-retinoic acid

Replacement therapy give platelets, fresh frozen plasma (FFP), cryoprecipitate

o Heparin may be useful in acrocyanosis, thrombophlebitis, etc

Microangiopathic Hemolytic Anemia

Blood smear schistocytes and microcytes (spherocytes), accompanied by thrombocytopenia

Intravascular hemolysis caused by red cells traversing small blood vessels with fibrin
deposition or platelet aggregates or in areas of high turbulence

Fragmentation fragments are targeted for destruction in RES (spleen)

DDx DIC, TTP, HUS, malignant HTN, aortic stenosis, HELLP syndrome/eclampsia, HIT, severe
GN
Thrombotic Thrombocytopenic Purpura

Classic pentad MHA (see above), thrombocytopenia, renal involvment, neuro signs, fever

Pathogenesis autoantibodies inhibit ADAMTS13 (metalloprotease vWF cleaver in platelet-rich

thrombi) large amounts of platletsaccumulate at clot (congenital deficiency = UpshawSchulman syndrome)

Labs hemolysis, thrombocytopenia, abnormal renal tests

Treatment plasma exchange +/- immunosuppression

Vitamin K Deficiency

Vitamin K functions to carboxylate a number of coagulation enzymes & inhibitors to active

form

Vitamin K deficient coagulation enzymes that have been produced are no longer active

Etiology includes nutritional depletion, antibiotic SE, and warfarin effect:

Nutritional depletion alchoholics, long-term TPN nutrition no more vitamin K

Antibiotic administration can interfere with bacteria making vitamin K in gut

Warfarin inhibits epoxide reductase (which recycles vitamin K)

Liver Disease

Liver function to synthesize and clear both procoagulants and inhibitors

Factor VIII usually increased (due to lack of clearance)

Factor V will be reduced; distinguishes from vitamin K deficiency

Fibrinogen very last enzyme to fall; signal of severe liver disease

Liver disease sequelae portal HTN, varices, gastritis, hemorrhoids bleeding

Massive Transfusion

Massive Transfusion transfusion of more than 1.5x patient blood volume in 1 day

Acquired coagulopathy from dilution of plasma & platelets with excess

anticoagulant hemorrhage

Anticoagulant about 10% of a transfusion is anticoagulant can build up easily in

blood

Prevention administer 1 unit FFP & calcium chloride for every 4-6 units of PRBCs (packed
RBCs)
Prolonged aPTT, normal PT

Factor VIII inhibition antibody meadiated; will affect intrinsic pathway only

Etiologies can be elderly, post-partum, CT disorder, or B cell malignancy

Clinical see ecchymoses, hematomas; favorable prognosis

Tx give immunosuppressive Tx, also give clotting factors during bleeding as bypassing
agents (activated prothrombin complex concentrates, recombinant factor VIIa)

16 PLATELET DISORDERS
Thrombocytopenia of Decreased Platelet Production

Bone Marrow Disease include 1o failure, invasion, or injury:

o 1o bone marrow failure bone marrow idiopathically stops producing
blood cells
o Bone marrow invasion from metastatic cancer, myelofibrosis, or
cancer in situ
o Bone marrow injury reaction to drugs, radiation, chemicals,
alcohol
Nutritional Disorders leading to lack of compounds necessary to build
platelets
Megaloblastic Anemia Fe deficiency affects platelet production
Hereditary Disorder involve decreasing megakaryocytes or
producing bad megakaryocytes
Immune Thrombocytopenias

Autoimmune thrombocytopenia antibodies produced against platelets:

o Acute more common in children, preceded by viral infection;
generally self-limited
o Chronic commonly seen in women 20-40y, a chronic disorder,
have normal bone marrow
Tx give immunosuppressives, reduce platelet removal by
macrophages
o Secondary associated with disordered lymphoid function (e.g. SLE,
lymphoma, leukemia)
Post-transfusion Purpura patients serum contains antibodies to platelet
antigens of donor blood
o Pla1 most common antigen on a platelet which can have antibody
response
o Innocent bystander mechanism often patients own platelets also
destroyed in Ig response
Neonatal Isoimmune Thrombocytopenia maternal antibodies to neonate
platelets transferred in utero

Mother is Pla1 negative, and has been previously sensitized to

Pla1 develops Igs
o Neonate is Pla1 positive, and mothers transferred serum mounts
immune response
Drug-Induced Immune Thrombocytopenia

Common Drugs include sulfa drugs, penicillin, gold salts, dilantin, lasix
Mechanism several different mechanisms drugs cause immune reaction:
o (Comp. Hapten Mech) drug binds to platelet, Ig recognizes,
platelet bystander destroyed
o (Protein/Drug Complex) drug binds to platelet
surface sturcture complex Ig attacks
o (Comp. In Vivo Sensitization) drug binds to platelet surface
antigen neo-antigen Ig
Treatment REMOVE DRUG!!!
Thrombosis problem Ig response invokes complement system on
platelets, pro-thrombotic contents
lysed into blood

Heparin-Induced Thrombocytopenia

Mild direct chemical interaction between heparin & platelet

surface; premature clearance, rarely <100K
Severe life-threatening; several stages:
1) Heparin & platelet factor IV combine (Hep-PF4)
2) IgG antibody binds to Hep-PF4 to form immune complex
3) Fc region of antibody binds to platelet, and platelet activates
4) Large quantities of PF4 released into plasma
5) PF4 will bind to more heparin, or heparin-like proteins (heparans)
on endothelium
6) Immune response damages endothelium limb ischemia,
myocardial infarction, stroke
Non-Immune Thrombocytopenias

Dissmeinated Intravascular Coagulation coagulation uses up all

available platelets
Thrombotic Thrombocytopenic Purpura (TTP) disorder
of vWF proteolysis
o Mechanism lack of ADAMTS 13, cleaves long vWF; without =
massive platelet plugging
o No Fibrin vWF platelet plugs dont have fibrin (no clot cascade);
fibrin = DIC
o Findings include a classic pentad:
Microagniopathic hemolytic anemia
shows schistocytes on blood smear
Thombocytopenia platelets reduced
Neurologic dysfunction confusion
Renal Failure cant filter RBCs? Hemolytic uremic
syndrome
Fever also present
o Prevalence rare (1/250,000 each year, young adult females; 90%
fatal no Tx, <20% fatal w/ plasmpheresis

Splenic Sequestration splenic enlargement will cause spleen to hold up to

90% of platelets, rather than 20%
Thrombocytosis

Primary Thrombocytosis series of mutations leading to excess (often

malformed) platelets; rare
Secondary Thrombocytosis in response to inflammation, hemorrhage,
post-splenectomy, Fe def.
o Inflammation body produces blood cells to fight infection
o Hemorrhage makes sense for body to produce excess clotting
factors to stop bleeding
o Post-splenectomy no buffer for platelet storage
o Iron deficiency can result in an increase in platelet count
Thrombocytopenia Ranges

100,000 slight risk of bleeding

20,000-70,000 increased risk of bleeding with trauma/surgery
<20,000 increased risk of spontaneous hemorrhage
<10,000 increased risk of spontaneous intracranial hemorrhage
Platelet Function Tests

Peripheral Smear Are platelets visible? Size? Granules?

Platelet Count how many platelets are in the blood?
Bleeding Time highly variable; normal < 9 minutes
Platelet Aggregation/Secretion Studies assess platelet responses to
various chemicals
Congenital Qualitative Platelet Disorder

Qualitiative Platelet Disorder dysfunctional platelets; usually normal

count, variable bleeding
Congenital Platelet Disorders involve defects in adhesion, aggregation,
activation, and secretion:
o Adhesion Disorders vWF & GpIb interaction defective:
Von Willebrands Disease AD; vWF dysfunctional, cant
bind GpIb
Bernard Soulier syndrome AR; GpIb dysfunctional; cant
have vWF bind
Lab Results aggregation normal, just cant
adhere; flat ristocetin test; otherwise norm
o Aggregation Disorders defects in fibrinogen & IIb/IIIa receptor
Afibrinogenemia fibrinogen not available for binding
platelet IIb/IIIa receptor
Glanzmanns thrombocytopenia platelets
lack IIb/IIIa receptor
Lab Results adhesion normal (ristocetin normal), but flat
aggregation tests (ADP, Epi)
o Activation/Secretion Disorders platelets cant activate to
speculated form
Storage Pool Deficiency deficiency of dense/alpha granules
activating platelet

Stimulus-Response Pathway Defect defect in pathway

leading to granule secretion
Primary Secretion Defects defect in ability of platelet to
secrete granule after signal
Platelet Aggregation Pathway

1.
2.
3.
4.
5.

Stimulus binds to receptor

Receptor causes PLA2 to produce arachidonic acid
Arachidonic acid cascade produces thromboxanes
Thromboxanes cause release of fibrinogen granules
Fibrinogen binds two platelets together at IIbIIa receptor
Acquired Qualitiative Platelet Disorder

Uremia causes abnormalities in vessel wall &

platelet prostaglandin pathways Tx dialysis
Myeloproliferative Disorders loss of first and second wave of
aggregation arterial and venous thrombosis
Myelodysplasia, Leukemia usually produces bad platelets
Dysproteinemias increased plasma viscocity interferes with fibrin
polymerization, cant clot
Cardiopulmonary bypass tubing can partially activate platelets
Anti-platelet antibodies can bind & block receptors important for
adhesion & aggregation; generally have better platelet function than other
disorders here
Liver Disease bad protein synthesis coagulopathy; creates bad platelets
Drugs can bind to platelet receptors, interfere with action

17 THROMBOSIS

Virchows Triad includes stasis, injury, hypercoagulability:

o Venous Stasis slowed rate of blood fow; through immobility or
compression
o Vascular Injury involves damage to vasculature favoring deposition
of platelets/fibrin
o Hypercoagulability blood components are in favor of thrombosis to
begin with
Related disorders thrombophlebitis, compartment syndrome, DVT
Venous Thrombosis Risk Factors >40, obese, Hx, stasis, surgery,
parturition (clot to stop childbirth
hemorrhage), estrogen, medical
disorders, genetic thrombophilia
Genetic Thrombophilia indicated by thrombosis <50 yo, recurrent
thromboses, Fam Hx, and
thrombosis @ unusual site (portal/renal vein,
mesenteric, CNS venous sinus)

Congenital Anticoagulant Deficiencies

Protein C Deficiency vitamin K dependent protein, inactivates Va, VIIIa

o Prevalence present in 1 in 200-500 individuals, 2-9% of individuals
w/ venous thrombosis
o Vs. Acquired many diseases lead to acquired Protein C deficiency,
so be wary of Dx congen.
o Warfarin (Coumadin) will lower protein C levels, so
cant Dx when on these drugs
Warfarin hypercoagulability warfarin dec. protein C faster
than coagulants initially!
Protein S Deficiency acts as a cofactor for Protein C
o Free Active Form only 40% circulates in this form, rest is bound
by C4b
o Vs. Acquired same as Protein C many diseases lead to this, may
not be congenital
o Oral contraceptives, pregnancy will lower protein S levels, so
cant Dx at these times
Antithrombin III Deficiency binds natural heparin-like molecules to
activate & anti-coagulate
o Action binds & inhibits thrombin & Xa, along with IXa, XIa, XIIa
o Severity much more severe than protein C/S deficiencies; causes
very early thromboses (teen)
o aPTT test heparin wont prolong, since ATIII isnt really there!
Often how Dx is made
o Reduced ATIII can be lowered by chronic heparin therapy, or acute
thrombosis
Pro-Coagulant Thrombophilia

Factor V Leiden mutation in Factor V leading to resistance to protein

C hypercoagulability
o Protein C Resistance protein C can no longer deactivate mutated
factor V
o Prevalence only Caucasians
o Thrombosis 7x increase in risk
o Oral contraceptives will magnify thrombosis risk
Prothrombin 20210 Mutation excessive prothrombin synthesis
o Thrombosis also increased risk
o Prevalence also only in Caucasians (some SE Asians too, likely
result of trade routes)
Fibrinolysis Abnormality decreased levels of plasminogen or elevated
plasminogen inhibitor (PAI-1)
Hyperhomocysteinemia associated with premature atherosclerosis,
venous thromboembolism
o Oxidative damage elevated levels of homocysteine lead to vascular
damage thrombosis
o CAD greater risk of CAD due to accelerated atherosclerosis
o Methylene Tetrahydrofolate Reductase elevated MTHFR leads to
elevated homocysteine
Dysfibrinogens fibrinogens polymerize too easily, or which lack
cleavage sites thombosis
Excessive Clotting Factor most commonly too much Factor
VIII hypercoagulability
Arterial/Venous Thrombosis

Arterial Thrombosis atherosclerotic plaques, a white thrombus

o Elevated lipoproteins, factor VII, homocysteine,
platelets, GpIIbIIIa gain-of-function
Venous Thrombosis a red thrombus
Lipoprotein (a)

Lp(a) modified LDL, serum levels are genetically determined

Structure homologous to plasminogen interfere w/ fibrinolysis and
interacts with macrophages to promote foam cell formation and deposition of
cholesterol
ICAM-1 enhanced expression leading to recruitment and binding of
monocytes to vessel wall
Levels associated w/ cerebrovascular disease in men

18 PEDIATRIC HEMATOLOGY
Embryonic Hematopoiesis

Yolk Sac hematopoiesis site during first trimester; begins 15-20 days,
ends 12 weeks
Liver hematopoiesis site during second trimester; stem cells from yolk
sac migrate here; ends at birth
Bone Marrow Space 1o hematopoiesis site during third trimester and
beyond; space formed by 3 mos, cellular by 20 wks, primary site by 24 wks
Hemoglobin Synthesis

Chromosomal distribution 11 has genes for beta chain, 16 has genes

for alpha chain
Gower 1 & 2 earliest types of hemoglobin 1st trimester, use 22 and
22 subunits
Fetal another hemoglobin type, made in liver, uses 22 subunits
A1 earlier main adult hemoglobin type, made in marrow,
uses 22 subunits
A2 final supplementary adult hemoglobin type, made in marrow,
uses 22 subunits
Hemoglobin Values in Newborns

Neonate has high (17-18) hemoglobin concentration (from both fetal &
adult hemoglobin made)
2 Months has low (10-12) hemoglobin concentration (from fetal
hemoglobin stopping) physiologic anemia
Child 1-12y has slightly low (12-13) hemoglobin conc.
Adult has normal (14-16) hemoglobin conc.
Retic count decreases from 28 wks onward
Newborn RBCs

Rigidity neonate eggshell RBCs are more rigid and less

permeable than adult RBCs
Stability neonate RBCs are less stable, w/ more unstable
methemoglobin, & more denaturation-prone
O2 Affinity neonate RBCs have greater O2 affinity (fetal Hgb)
Well water has high nitrite/nitrate concentrations,
oxidizes Hgb methemoglobin; dont give to babies
Clamping strategy because 1/3rd of blood volume in placenta at time of
birth, OB/GYN holds newborn at level of womb for 2 minutes then
clamps; clamp early and child is anemic, clamp late and child
is polycythemic
Neonate Platelets

Premature Neonates have low platelet counts (220,000 350,000)

Normal Neonates have high platelet counts (250,000 380,000)
Normal Adult/Child normal platelet counts (250,000 350,000)
Always abnormal despite low premie platelet counts, a count less than
150,000 = always abnormal
Neonate Platelet Function equivalent to adults
Newborn WBC

Neonate have a very high PMN count, helps protect against infections
during birth
Infant-Preschool PMN count dips down, and high lymphocyte count,
helps develop immunity
School-age-Adult lymphocyte count dips back to normal level, PMN count
picks back up to normal
Newborn Coagulation Factors

Clotting Factors diminished synthesis & high clearance during birth, but
o Vitamin K Dependent Factors Factors 2, 7, 9, 10 all diminished at
birth vitamin K supp.
o PT, aPTT both elevated (lack of clotting) at birth
o Factor VIII should be normal at birth, so if low Hemophilia A
o Factor IX vit K dependent, so cannot diagnose Hemophilia B at birth
Anti-clotting Factors (Protein C/S) even more diminished than clotting
factors, thus thrombosis risk
Newborn Diseases infections of newborn (rubella, CMV, toxo, syphillis, malaria)
can lead to hemolytic anemia & thrombocytopenia
Newborn Iron normal @ full-term (takes iron from mother, even if mom low),
but low in premies (get iron late); thus if <36 wks at birth, iron supplementation is
necessary
Erythroblastosis Fetalis

Erythroblastosis Fetalis left-shift of RBCs (nucleated RBCs) in neonates,

from hemolytic anemia
Pathogenesis most commonly from Rh antigen on RBCs mother
produces antibodies hemolysis
o Transfer only need about 1 mL of fetal RBC to be transferred to
elicit maternal immunity

ABO incompatible if mother & child also ABO

incompatible risk reduced of EF!, since this immune response
quicker than Rh response, but is primarily an IgM response, which
cant cross placenta (IgG only = Rh response)
o Obstetric Procedures C-section, amniocenesis, etc have risk of
blood transfer increase risk for next child
Compensation infant forced to increase erythropoiesis (leads to leftshift RBCs) so they are only mildly anemic at birth
Sx include jaundice, pallor, hepatosplenomegaly, hydrops:
o Jaundice seen quickly after birth, from hyperbilirubinemia due to
hemoglobin breakdown
o Pallor due to anemia of hemolysis
o Hepatosplenomegaly (HSM) due to liver overdrive to produce
compensatory RBCs
o Hydrops CHF due to heart recognizing anemia & pumping way too
fast
Lab Dx low hemoglobin, high retics/nucleated RBCs, hyperbilirubinemia
Tx force an early delivery, before CHF hydrops; can also give intrauterine
transfusions
o Exchange transfusion at birth, put catheter into fetal blood line &
replace with compatible
RBCs which wont lyse from transferred maternal
antibodies if Hgb < 12 or Bilirubin >20

Maternal plasmapheresis get rid of maternal antibodies

produced
Prevention can use Rhogam antibodies against Rh, given to mother so
that this fake immune
response can prevent the development of a real
immune response

19
20 MYELOID CELL DISORDERS
Myeloid Cell Maturation

GM-CSF granulocyte-m-phage col. stim. factor; myeloid stem

cell (myeloblast)/(immature monocyte)
G-CSF granulocyte colony stimulating factor;
stimulates myeloblasts N. promyelocyte
Use as drugs both GM and GCSF are in use as drugs to cause
differentiation in neutropenias

WBC

WBC Measurement uses automated counter, refects circulating pool

of myeloid/lymphoid cells
Relative/Absolute Amounts each WBC type indicated by relative (%)
and absolute (% * WBC)

Infant WBC usually very high WBC (fighting infections), high

PMN compared to lymphocytes
Child WBC also somewhat high WBC (developing immunity), high
lymphocytes compared to PMNs
Adult WBC has a lower WBC (mature immune system), high
PMN compared to lymphocytes
Disease States can also affect WBC:
o Bacterial infection huge increase in WBC, high PMN % and leftshift (band cells)
o Steroid therapy actually increases WBC, due to high PMN
% (marginating effect)
o Splenectomy also increases WBC, due to putting more into active
circulation, less sequestered
o Viral infection actually decreases WBC, due to suppressive
effect of virus on bone marrow
o Chemotherapy has huge decrease in WBC, although monocyte
increase after recovery
Neutrophil Maturation

Proliferation
involves myeloblast N. promyelocyte N. myelocyte in
bone marrow (6-7 days, 25%)
Maturation longest, involves N. myelocyte N. metamyelocyte N.
band PMN in marrow (6-7 days, 65%)
Intravascular/Tissues about 10% of neutrophils finish development
here, N. band PMN
Neutrophilia

Neutrophilia an abnormally high PMN count (>7700), often involving

a left-shift (more bands/precursors)
Types either an acute shift or a chronic stimulation:
o Acute shift a sudden shift of PMNs
from marginating circulating pool, (not a total WBC )
Causes include steroids, exercise, epinephrine, hypoxia,
seizures, stress
o Chronic stimulation excess cytokines stimulating proliferative pool;
a real WBC
Causes include infection, pregnancy, chemo recovery
Disorders causes are Downs
syndrome, myeloproliferative dz., marrow metastases
Neutropenia

Neutropenia an abnormally low PMN count (<1500), with risk of

infection
o 500-1000 infection from exposure
o <500 infection from host organisms
o AfAm lower normal nphil counts (1000-1200)
Types include decreased production, increased destruction, or shift
to marginating pool
o Acute shift a sudden shift of PMNs
from circulating marginating pool (not a real WBC )

Infection a severe infection (penetrates to

tissues), endotoxin release will cause
Medical procedures artificial circulations, such
as hemodialysis, cardiopulm. bypass
o Decreased production fewer PMNs produced in bone marrow
Immunosuppression some medications, chemotherapy,
antibiotics will cause
o Increased destruction more PMNs destroyed in peripheral
circulation
Autoimmune diseases such as RA, SLE will cause
Management any time patient has fever + neutropenia (F+N), good
reason to hospitalize
o Medications many medications have neutropenia as a known SE;
look through these
o Bone marrow look for malignancies in bone marrow which indicate
myeloproliferative disorder
o Cyclic neutropenia inherited disorder (auto dom) where every 3
weeks patient has neutropenia (fever, mouth ulcers); treat w/ G-CSF,
usually improves after puberty
o Congenital neutropenia PMN maturation arrest; freq.
infections, mouth sores, leukemia risk
Tx give artificial G-CSF, bone marrow transplant (BMT)
Neutrophil Function

Response Process when tissue is damaged, chemotactic

factors released which induce PMN response:
1) Rolling & attachment PMNs stick to endothelial cell wall due to
chemotactic interactions
2) Adhesion integrins allow for tight adhesion of PMN to vessel wall
3) Diapedesis margination of PMN across endothelial cell wall
4) Phagocytosis PMN phagocytosis offending organism, contains
within lysosomal vesicle
5) Granule release granules, along with peroxide & superoxide
released to kill organism
PMN disorders can involve a disruption in function at any step of response
process above:
1) Sialyl LewisX PMNs lack protein needed for rolling/attachment
2) LAD-1 a leukocytic adhesion protein (integrin) defect (Type 1),
PMNs cant adhere
3) -4) -5) Chediak-Higashi Syndrome (CHS) defect in granule
formation (too large); associated w/ oculocutaneous albinsim,
neuropathy, frequent infxn; treat w/ bone marrow x-plant
Chronic Granulomatrous Disease defect in peroxide/superoxide
formation
Catalase (): CGD can kill Cat () bacteria (strep B), since they
produce own peroxide
Catalase (+): CGD cant kill Cat (+) bacteria (E. coli), since
they break down peroxide
Myeloperoxidase Deficiency more common, defect
in hyperchlorous acid (HOCl), usually

clinically silent, although bacterial killing

takes longer than norm
Monocytes/Macrophages

RES the reticuloendothelial system is the monocyte/macrophage system

Maturation again involves proliferation, maturation (shorter),
and intravascular/tissue (longer)
Function can phagocytose foreign particles after nphils arrive at site
and release cytokines to attract them
o Destruction can destroy phagocytosed particles (innate immunity)
o T-cell presentation APCs can also present phagocytosed particles
to T-cells (adaptive)
Defects defects of PMNs also affect monocytes (LAD, CHS, CGD)
Monocytopenia low monocyte count, can occur with steroids or stress
Monocytophilia high monocyte count, can occur with tumor, infection,
CGD, marrow recovery and a number of infections (malaria, TB,
RMSF, leshmaniasis, brucellosis, mononucleosis)
Eosinophils

Maturation also have a faster maturation than PMNs

Function bright red granules with IgE on surface (unlike PMNs); key role
in killing parasites
Eosinophila elevated eosinophils (<400), NAACP conditions:
o Neoplasm hodgkins disease, lymphoma, other tumor
o Allergies induced by environment, drugs
o Asthma has elevated eosinophils too
o Collagen vascular disease includes vasculitis
o Parasite infection of parasite
Idiopathic Hypereosinophilia too high eosinophil
count organ dysfxn; Tx immunosuppress, antihist
Basophils & Mast Cells

Maturation basophil like PMN, mast cell very quick

Function largely unknown, possible defense against parasites
Low Basophil count from steroids, hypersensitivity
High Basophil count from allergies, infection, endocrinopathies,
myeloproliferative dz (CML) systemic mastocytosis (symptoms due to
excess histamine release)

21 BONE MARROW STEM CELL DISORDER

PATHOLOGY
Cytopenia Disorders

Aplastic Anemia (AA) caused by scarcity of hematopoietic stem

cells in bone marrow
o Pancytopenia most common symptom, due to lack of
adequate blood cell production (nphils <500, platelets <20,000,
retic < 50,000)

o Bone marrow hypocellularity Dx of aplastic anemia

o 1o Idiopathic AA approximately half of cases
o 2o AA due to drugs, radiation, marrow toxins
Myelodysplastic Syndrome (MDS) progenitor cells
w/ abnormal differentiation (and thus function)
o Cytopenia can have one, several, or all cell lineages down,
depending on stage affected
o Acute Myelocytic Leukemia MDS progression
if myeloblasts spill into blood to level >20%
o Refractory anemia/cytopenia different subtypes of MDS
o Diagnosis made through lab smears, cytogenics, and
exclusion of nutritional disorders:
Lab Dx made thru blood & marrow
smears dysplasia, high blasts
Cytogenetics genotype to assess for clonality
Exclude folate/B12 deficiency can mimic MDS
o Histological Findings ringed sideroblasts, dysmorphic
granulocytes/megakaryoctes/platelets
Dysmorphic erythroid precursors nuclear
irregularity, weird granularity
Ringed sideroblasts blasts have trouble handling
Fe accumulate in ring around cell
Dysmorphic
granulocytes hypogranular, hyposegmented nuclei
Dysmorphic megakaryocytes two small nuclei
(multinuclear = normal)
Dysmorphic platelets giant, hypogranular
Other Cytopenia Causes include infection, DIC, hypersplenism,
marrow toxins, nutritional, tumors
Chronic Myeloproliferative Disorders (CMPD)

CMPD involves progenitor cells with unregulated

growth but normal differentiation (at least initially)
o Elevated peripheral counts unlike MDS cytopenia, CMPD
presents w/ elevated counts
o No dysplasia in most cases, dysplasia is not prominent
o Vs. Reactive proliferation these occur on reactive basis;
CMPD occurs on neoplastic basis
Leukemoid Reaction elevated WBC count in response
to physiologic stress/infection
Toxic granulation neutrophils will granulate in
mounting active immune response
Dohle bodies very dark
blue blotches stacks of RER (revved up to fight
infection)
Normal cytogenetics no preponderance of a
clonal progenitor cell
Secondary Polycythemia hypoxic patient (smoker,
high altitude) will generate more cells
Reactive thrombocytosis more platelets due to
surgery, infection, hyposplenism, or hemorrhage
Mimic CMPD a reactive proliferation must be
distinguished from a CMPD

o Vs. Acute leukemia AL has very increased proportion of

blasts, rapidly fatal if no Tx
Chronic Myelogenous Leukemia (CML) blood & bone marrow
proliferation of myeloid cells
o Slight left shift earlier lineages present in blood,
but few blasts (mostly normal differentiation)
o Basophilia unsure why, but high basophil count distinctive
characteristic of CML
o Philadelphia chromosome virtually all CMLs have
this genetic marker (balanced translocation of part of 22 to 9)
or BCR/ABL fusion
o Granulocyte dominance CMLs affect multipotent stem cells,
but granulocyte lineage dominant
o Accelerated Phase if blasts in blood are 10-19%
o Blast Crisis if over 20% blasts in blood; 2/3 myeloid, 1/3
lymphoid multipotent stem cells
Polycthemia Vera (PV) bone marrow disease producing
primarily excess RBCs (also WBCs/platelets)
o Erythroid dominance although affecting multipotent stem
cells, erythroid lineage dominates
o Normal O2 saturation unlike 2o polycythemia reactive
proliferation caused by hypoxia
o Decreased EPO low erythropoietin level due to feedback,
unlike 2o polycthemia (elevated EPO from hypoxia)
o Thrombotic/hemorrhagic complications due to increased
viscosity & platelet dysfunction
o JAK2/V617F mutated in nearly all cases; late phase nearly
indistinguishable from
Chronic Idiopathic myelofibrosis disorder of displaced
proliferation (bone marrow hypocellular)
o Displaced proliferation spleen and liver take over
hematopoiesis processes (extramedullary)
o Bone marrow progressively hypocellular & fibrotic
o Bone thickening abnormally thickened bony trabeculae
o Splenomegaly gross enlargment due to hematopoiesis in
liver and spleen
o Leukoerythroblastic peripheral blood smear left shifted
blood smear with:
Teardrop erythrocytes from having to migrate
through liver & splenic sinuses
Myeloblasts from left-shift, lack of marrow maturation
environment
o JAK2/V617F mutated in about 50% of cases
Essential thrombocythemia sustained platelet count > 600,000
o Exclusion Diagnosis must exclude other CMPDs:
Not reactive thrombocytosis no infection, Fe
deficiency, malignancy, or splenectomy
No RBC mass unlike polycthemia vera
No Philadelphia Chromosome not a CML
No marrow fibrosis unlike chronic idiopathic
myelofibrosis
JAK2/V617F mutated in 50% of cases but if detected
then only need count >450k to diagnose

o Abnormal megakaryocytes numerous abnormal

megakaryocytes in bone marrow
o Dysfunctional platelets numerous platelets are
morphologically bizarre & dysfunctional
o Thrombosis/hemorrhage due to large numbers of
dysfunctional platelets
Acute Leukemia

Acute Leukemia Criteria blast content of blood must be >20%

o Bone marrow largely replaced with blast forms that remain
undifferentiated
o Elevated WBC leukemic blasts in the blood may cause this
o Leukemic infiltration leukemic cells may infiltrate liver,
spleen, lymph nodes, skin, etc.
Lab Dx based on CBC differential, bone marrow & blood smears
o CBC can show anemia, neutropenia, thrombocytopenia
from blast forms only, few mature cells
o Lymphocytic/Myelogenous use cytochemistry, flow
cytometry, cytogenetics to subclassify
Myeloperoxidase evidence of granulocytic
differentiation
Non-specific esterase evidence
of monocytic differentiation
o Hemorhage/thrombosis can also be caused by acute
leukemia
Acute Lymphocytic Leukemia (ALL) leukemia
of lymphoblasts (>20%)
o Children Prevalence ALL much more common in children
than adults (think hi lymphocytes)
o B/T subtypes can assess B/T lineages thru fow cytometry;
may have prognostic significance
o Philadelphia chromosome has bad prognosis if cytogenetics
confirms this
o Bone marrow biopsy shows montonous accumulation of all
blasts
o Peripheral smear will show blasts in smear
Acute Myeloblastic Leukemia (AML) leukemia
of myeloblasts (>20%)
o Adult Prevalence AML much more common in adults (think
lower lymphocytes)
o Azurophilic cytoplasmic granules myeloblasts have these,
unlike lymphoblasts
o Auer rods crystalline structure in cytoplasm formed by
myeloperoxidase Dx AML
o Subtyping several subtypes of AML,
with prognostic/therapeutic significance
M3-acute promyelocyte leukemia will see multiple Auer
rods, heavy granulation of promyelocytes, risk of
DIC,Tx retinoic acid, Ch 15-17 translocate
Laboratory Dx of Acute Leukemias
1)

Smear morphology & cytochemistry accurate classification of

majority of acute leukemias
Myeloperoxidase granulocytic differentiation

 Nonspecific esterase monocytic differentiation

Immunophenotyping (flow cytometry) confirms Dx of ALL; may
be needed in some AMLs
3) Cytogenetics/molecular genetics important supplemental
prognostic & Dx information
2)

22 ACUTE LEUKEMIA
Acute Myeloid Leukemia Presentation (Leukostasis, Infiltration, DIC), Labs

AML acquired genetic defect of unregulated myeloid growth w/ limited

or no differentiation
Presentation shows symptoms of bone marrow failure:
o Leukostasis increased viscosity of blood
with pulmonary/cerebral effects (SOB at rest)
o Extramedullary disease myeloblastomas
o Infiltration leukemic infiltration of skin, gingiva, liver, spleen, lymph
nodes, CNS
o DIC from increased viscosity & platelet dysfunction
Lab Dx patient will have hyperuricemia, hypokalemia, and spurious
hypoxemia

AML

Etiology can be a part of a syndrome, or reactive from an insult:

o Radiation, toxins (benzene) can induce an AML state
o Treatment-related alkylating agents (cyclophosphamide),
topoisomerase II inhibitors
o Congenital DNA Repair Disorders Fanconis syndrome, Blooms
syndrome
o Myeloproliferative & Myelodysplastic Disorders, Aplastic
Anemia can progress to AML
o Treatment related alkylating agents (5-7 yrs later), topoisomerase
II inhibitors (1-2 years later)
AML Prognosis include age, blast karyotype, MDR phenotype, patient Hx,
molecular indicators
o Age although common in children, increased incidence & severity
w/ age
o Performance Status if patient feels bad, disease prognosis is bad
o Karyotype certain karyotypes good, others bad prognosis:
t(15;17) karyotype with good prognosis; associated
w/ acute promyelocytic leukemia
t(8;21), inv(16) other karyotypes with good prognosis
(better remission)
5del, 7del, Phl chromosome poor prognosis
Core binding factors (CBF and -) TFs for blood cell
production, lost in t(15;17)

Multi-Drug Resistant (MDR) Phenotype have toxin

pumps cant treat as easily!
AML Molecular Prognosis mutations in FLT3 receptor can make for bad
prognosis
o FLT3 Activation mutations in areas of gene will activate receptor
o Function receptor binds ligand and signals cell
growth high WBC
o Prognosis associated with a worse outcome, and remains negative
prognostic factor following HSCT
o Karyotype associated with normal karyotypes
Nucleophosmin1 mutation results in better prognosis
o Mutation frameshift mutation results in nuclear export signal so
protein goes to cytoplasm
o Prognosis in younger patients with normal cytogenetics, NPM1
mutation predicts favorable prognosis
AML Treatment

Induction induce a complete remission of AML through combination

chemotherapy
o Leukemic blast clearance remove all leukemic blasts by
morphologic review
o Anthracycline + Ara-Cytarbine wipe out marrow (aplasia),
an induction chemotherapeutic
o G-CSF Priming give simultaneously w/ Ara-C, induce cell
division (Ara-C only kills dividing)
Bad idea priming hasnt proven to be helpful except young
pts w/ normal karyotype
o MDR Modulation stop MDR pump from working, but more toxicity
elsewhere (bad idea)
o Intense Ara-C higher doses better in children, who can tolerate
more
Post-Remission Therapy weigh issues; maintain remission & replenish
blood cells:
o More chemotherapy maintain remission, make sure few remaining
leukemic cells dont resurge
o Bone marrow transplant need to replenish blood cells eventually,
treatment of choice in peds
o Age young patients easier to get into remission, can take more
chemo
AML Biologically Targeted Tx

Acute Promyelocytic Leukemia occurs in younger patients, associated

with biologically targeted Tx:
o Histology leukocytes cells are hypergranular with isthmusshaped nuclei, Auer rods
o Genetics involves t(15;17) fusion protein
o Treatment fusion protein can respond to large doses of retinoic
acid combined w/ chemo
SEs capillary leak syndrome (wt gain,
hypoxia, pleuropericardial effusions, lung infiltrates, fever);
onset day 2 to week 3,hyperluekocytosis, rapidly fatal; treat w/
dexamethasone
o Prognosis targeting with RA makes for good prognosis

Relapse treat with Arsenic trioxide also moderately successful

(same types of SEs as ATRA)
CD33 cell surface protein on AML blast cells, spans membrane
o CD33 Antibody gemtuzumab antibody against CD33 protein can
help target AML blasts
o Toxicity infusion-related rxn, myelosuppression, hepatotoxic,
hepatic veno-occulusive dz
o Remission helps get disease in remission, although not curative Tx\
o High risk mutations didnt help with high-risk patients
FLT3 protein with gain-of-function mutation which induces blast
proliferation bad AML prognosis
Lestaurtinib an oral FLT3 inhibitor, can be synergistic with
chemotherapy Tx
o Sensitivity some patients are sensitive to FLT3
inhibitor remission; some arent no remit

ALL

Acute Lymphoid Leukemia much less common in adults; more

common in children
Prognosis determined by age, cytogenetics, immunophenotype:
o Age children 2-9 yo have best prognosis, because like to
have t(12;21)
o Cytogenetics t(12;21) = good prognosis
o Philadelphia Chromosome can be present in adults w/ ALL
(not just CML), poor prognosis
o Immunophenotype those affecting T cell lineage = better
prognosis (than B cell lineage)
Treatment give an extended induction & post-remission treatment
o Induction give anthracycline plus steroids, vincristine 2-3 years
o Post-Remission chemotherapy is prolonged compared to AML;
eventually allogenic transplant
o CNS prophylaxis ALL likes to lie dormant in CNS, need to prevent
relapse here
Outcomes 80-90% can go into remission, but survival poor 30% adults,
70% children; HSCT is recommended in adults w/ high risk features or relapse

23 LYMPHOMA PATHOLOGY
Lymphoma

Lymphoma a malignant neoplasm of cell derived

from lymphocytes/lymphocyte precursors
Sites where lymphocytes typically reside (lymph nodes, spleen, etc.), but
can be basically anywhere
o BALT bronchus-associated lymphoid tissues
o MALT/GALT mucosa/gut-associated lymphoid tissues (mostly same
thing)

o Other key sites skin, thymus, bone marrow, blood

vs. ALL in ALL, lymphocytes/blasts are in the circulation; in lymphoma,
they are in a palpable mass (with exceptions)
B-cells more common lymphoma problem in US than T-cells (this lecture
focuses on B-cells only)
Lymphoma Node Anatomy

Lymph Node Anatomy contain cortex, which has lymph node

follicles within, comprised of:
o Germinal center B-cell rich area of lymph node (antigen-stimulated
differentiation occurs)
o Mantle zone Cuff of dark cells around germinal center
o Marginal zone thinner layer around mantle zone, seen best in
spleen
Pre-follicular B-cells are nave recirculating B-cells, located in mantle
zone or blood; havent diffd
Follicular B-cells cells from mantle zone can travel deep into germinal
center differentiate to stim.
o Somatic gene mutation takes place inside germinal center; can be
used to identify follicular
Post-follicular B-cells already differentiated memory B-cells, located
in marginal zone or in elsewhere
Lymphoma Classification

Pre-follicular B-cell Lymphomas include CLL/SLL & mantle cell

lymphomas:
o Chronic Lymphocytic Leukemia/Small Lymphocytic Leukemia
(CLL/SLL) since pre-follicular B-cell lymphomas are in the circulation
often, they are a leukemia in this sense
Monoclonal CLL/SLL presents as excess lymphocytes in blood
of same size/shape
Caveat morphologically, CLL/SLL appears pre-follicular, but
molecular studies indicate some are actually post-follicular.Prefollicular subtypes have more aggressive Home
o Mantle Cell Lymphoma cells of mantle zone become neoplastic &
invade outside of boundary; more aggressive than CLL/SLL; irregularly
contoured cells
Follicular B-cell Lymphoma include follicular, large cell,
and Burkitts lymphoma
o Follicular lymphoma presents as many invading follicles within a
lymph node
Small cleaved cells if this cell type present in follicle, less
aggressive course
Large round cells more of this cell type present in
follicle, prognosis worse but responds to therapy well
because dividing faster
o Large cell lymphoma follicle only contains large round cells,
can expand out of germ. ctr.
Prognosis lymphoma is highly aggressive, but this
means Tx can at least work better
Treatment aims at killing dividing cells, so works better on
aggressive proliferating

Burkitts Lymphoma incredibly aggressive, increased number of

mitotic figures, often EBV association
Genetic Defect most commonly t(8;14)
Post-Follicular B-cell Lymphoma include marginal zone lymphoma (MALT)
& lymphoplasmacytic
o Marginal Zone lymphoma pale marginal zone is abnormally
expanded
Spleen, GI Tract MALT common sites of marginal zone
lymphomas
H. pylori infection by this bug can induce MALT lymphoma
o Lymphoplasmacytic lymphoma induces syndrome
of Waldenstroms macroglobulinemia
Plasma-cell prone since post-follicular B-cells have
differentiated already, some are predisposed to activate into
plasma cells lymphoplasmacytic
IgM secretion main initial Ig secreted vs. no IgM
in plasmocytoma
Waldenstroms macroglobulinemia thickened blood due
to high Ig secretion
Classification Scheme lymphoma subtypes are based upon analogy to
normal cell types in lymph node
o Morphology some basic lymphomas can be identified based on
morphology alone
o Surface marker studies most lymphomas are complex & require
cytometry/immunostain to ID
Pan-B CD19, CD20, CD22, CD79a
Pant-T CD2, CD3, CD4, CD7
CD5 normally found on T-cells but pathognomic of B cell
lymphomas
CD10, Bcl-6 marker of germinal center differentiation
Cyclin-D1 mantle cell lymphoma, some plasma
cell myelomas, some hairly cell leukemias
Specific patterns each lymphoma has
specific immunophenotypes that can be used to identify
o Molecular genetics some lymphomas are defined by chromosomal
abnormalities
Clinical Dx of Non-Hodgkins Lymphomas

Non-aggressive, but incurable CLL/SLL, follicular lymphoma, marginal

zone, lymphoplasmacytic
Aggressive, incurable Mantle cell lymphoma
Aggressive, potential cure Large cell lymphoma
Highly aggressive (potential cure) Burkitts lymphoma
Hodgkins Disease

Hodgkins Disease a clinically & histologically distinct type of lymphoma:

o Contiguous spread tends to spread contiguously from one lymph
node to the next
o Mediastinal mass common presentation of Hodgkins disease
o Inflammatory reaction histology can show nonneoplastic infammatory reaction to tumor cell

Reed-Sternberg cell/Hodgkin Cell neoplastic cell type in disease,

often surrounded by inflammation
o Histology can be bi-lobed nucleus, w/ 2 clear nuclei & 2 dark
nucleoli Owls eye
o What is it? a B-cell, but very distinctive shape & properties
Prognosis very responsive to therapy, often curable
Subtypes classical (nodular sclerosis, mixed cellularity, lymphocyte
depletion) & lymphocyte predomint.
o Classical Hodgkins Disease Nodular sclerosis most common; lymph nodes
have sclerotic nodules
Mixed cellularity another type
Lymphocyte depletion very rare
o Lymphocyte predominant Hodgkins Disease:
Totally different disease lymphocyte predominant different
from classical!
Behavior acts like a low-grade, slowly progressive nonHodgkins lymphoma

24 LYMPHOMA
Lymphoma

Lymphatic System network of vessels carrying lymph (w/ immune

cells) from tissues blood
Palpable Lymph Nodes based on features, can evince at normal, reactive,
or malignant Dx:
o Size smaller is more normal; (opp. malignant)
o Shape soft, flat, & fixed is normal; firm, spherical & movable is more
malignant
o Tenderness non-tender is normal; (opp. malignant)
o Stability non-growing is normal; (opp. malignant)
Sx can be asymptomatic, or notice lymph node changes & B-symptom
(constitutional) changes:
o Asymptomatic find lymphoma incidentally
o Lymph node changes usually painless enlargement in
neck/axilla/groin
o B-Symptoms unexplained fever, drenching night sweats, 10%
weight loss over 3 months
o Other constitutional changes fatigue and pain based on location
and size of mass
Dx can be made through lymph node biopsy, or if still unsure, a bone
marrow biopsy
Lymphoma: Hodgkins vs. Non-Hodgkins

Hodgkins Lymphoma has 3-4 main subtypes, 3 treated the same and
have same prognosis
o Prevalence more rare; about 3 times less common than NHL
o Demographic generally younger(20-29) patients, but also late peak
after 60 yo

o Spread predictable spread through contiguous lymph nodes

Non-Hodgkins Lymphoma many subtypes with varying
behavior/prognosis/Tx
o Prevalence 3 times more common than Hodgkins Disease
o Demographic incidence increases with age
o Spread more random spread
o Sites can involve Waldeyers ring, CNS, skin, GI tract/mesenteric
Lymphoma Staging Prognosis

Staging lymphomas (like any cancer) staged I-IV

o History & Physical assess for tumor size, nodal spread, metastases
o Labs can find cytopenias, organ dysfunction
o CT/PET Scans image lymph nodes
o Bone Marrow biopsy also assess for lymphoma
Diaphragm if lymph nodes on one side of diaphragm involved stages I/II;
if both stages III/IV; if patient has B symptoms then add B to stage; E
= extralymphatic, S = splenic
Hodgkins Disease Treatment

Limited (Early) Stage HD stages IA, IB, IIA; treated by:

o Chemotherapy + Radiation Therapy most common treatment
ABVD adriamycin,
bleomycin, vinblastive, dacarbazine general chemo
treatment
o Radiation Therapy Alone older method; irradiate lymph nodes
affected & adjacent ones
o Chemothearpy Alone debated for use among young; limits SEs of
radiation (particularly effects later in life)
o Prognosis good; 80-90% prevention of 5-year relapse
Advanced Stage HD stages IIB & up; treated by:
o Chemotherapy plays larger role, use longer treatment course
o Prognosis okay; about 2/3 cured (prevent 5-year relapse)
Salvaging Failed Treatments a number of options:
o Chemotherapy if radiation therapy was only given
previously, chemotherapy = 60% success
o High-dose chemotherapy + Autologous stem cell transplant if
previous chemo failed; 60%
o Post-transplant salvage very poor
Late Risks some of the complications of Tx now more concerning than HD
itself:
o 2o cancer, heart/lung damage common risks post-treatment
o Reduce/eliminate radiation proposed solution to reduce risks
Non-Hodgkins Lymphoma

Indolent, but incurable Follicular lymphoma, CLL/SLL, marginal

zone, lymphoplasmacytic
Aggressive, incurable Mantle cell lymphoma
Aggressive, curable Difuse large cell lymphoma
Highly aggressive, curable Burkitts lymphoma, lymphoblastic
(Italics = most common NHLs); 85% of NHL are B-cell
Indolent NHLs

Follicular lymphoma most common indolent lymphoma; many invading

follicles in lymph node
Marginal zone lymphoma (MALT) pale marginal zone of MALT is abnormally
expanded
Lymphoplasmacytic lymphoma memory B cells tend to activate into plasma
cells macroglobulinemia
Indolent NHL Prognosis & Treatment

Prognosis difficult treatment, but very slow progression

o FLIPI Follicular Lymphoma International Prognostic
Indicator predicts prognosis
Treatment Options observation, radiation, chemo, antibody,
transplantation:
o Watch & wait maintain good QOL while observing progression of
disease; treat when symptoms/complictation emerge; no difference in
survival whether treated immediately or observed; 2-3 years to
treatment
Pros avoids treatment toxicity, spontaneous regressions
occur, new treatments or new standards may become available
Cons requires periodic monitoring
o Radiation therapy good for early stage; knocks out lymph
nodes, but remission w/ adv. stage
o Chemotherapy ABVD?
o Antibody therapy such as Rituximab, monoclonal Igs against
surface markers (CD20)
CD20 surface marker present in Pre-B cell Activated Bcell, not plasm
Given with chem higher response rates and longer
remissions than chemo alone
o Radioactive antibodies bind to lymphoma, also irradiate nearby
lymphoma cells
Zevalin, Bexxar both radioactive antibody treatments for
indolent lymphomas
Regimen a single cycle treatment, lasts 1 week, two
infusions
Response great overall survival, also many without relapse
o Transplantation replace bone marrow
Aggressive Curable NHLs

Difuse Large B-cell Lymphoma most common type of lymphoma

Prognosis also scaled by various risk factors into a prognostic index
o Elderly worse prognosis, if chemo fails, many elderly cant withstand
bone marrow x-plant
o CNS inolvement risk bone marrow, sinuses, testes pose higher risk
of CNS involvement
Treatment should be initiated as soon as possible after diagnosis:
o Chemotherapy central role, CHOP chemotherapeutic combination
of drugs, 6-8 cycles
o Rituximab also part of standard Tx for B-cell lymphomas
o Low stage disease fewer cycles
o Salvage less than 20% of those who do not achieve CR with initial
therapy are cured but best option is autologous HSCT

Aggressive Incurable NHL

Mantle cell lymphoma pre-follicular cells of mantle zone become

neoplastic & invade outside boundary
Prognosis bleak; median survival of 3-4 years
Treatment initially give chemotherapy, but relapse & resistance high:
o Chemotherapy give CHOP chemotherapeutic combination of
drugs
o Remission short remission time; relapse rate high often resistant
to salvage regimens
Highly Aggressive Curable NHL

Burkitts lymphoma as well as lymphoblastic lymphoma, both

aggressive follicular lymphomas
Prognosis due to rapid division, can be easily targeted and very
responsive to chemo
Treatment give chemotherapy, also need to prophylax for CNS involvement
o Chemotherapy standard treatment
o CNS involvement has a high risk for this, need CNS
prophylaxis (intrathecal chemo and cranial radiation)
New Treatments

Stem Cell Transplants ways to make these safer, ways to prevent graft vs.
host disease
Vaccines also an new idea
New Drugs including proteasome inhibitors, histone deactylase inhibitors

25 CANCER PHARMACOLOGY
Cancer Pharmacology Principles

Behavior Modification change the behavior of malignant cells to become

non-malignant
o Retinoic Acid will induce differentiation of leukemic cells
in acute promyelocytic leukemia
Indirect Cell Killing create an environment in the host which will lead to
the killing of cancer cells:
o Bevacizumab antibody binds VEGF (vascular endothelial growth
factor), Tx colon cancer
Halted angiogenesis in colorectal cancer, tumor needs
neovascularization to grow
VEGF receptor endothelial cell binds VEGF, but cant if
VEGF complexed w/ Ig
o Rituximab antibody that binds to CD20 on B-cell lymphomas,
induces immune response to kill

Direct Cell Killing drugs which directly kill tumors:

o DNA damage drugs can induce DNA damage in tumor
cells apoptosis
o Antimetabolites drugs which prevent tumor cell from growing &
functioning
o Chromosome inhibitors drugs which inhibit the structure/function
of tumor chromosomes
o Survival inhibitors drugs which inhibit the survival signaling
pathways of tumor cells
Cancer Selectivity Strategies

Therapeutic Index goal of cancer Tx to widen, so just as valid to protect

host cells as to attack cancer
o Host Protection can help patient recover from lesions caused by
cancer/drug SEs:
Antiemetics keep patient from feeling nausea during
chemotherapy more chemo
Chemical lesions ifosfamide causes bladder
lesions protect with MESNA
Genetic lesions develop bone marrow factors which
is more resistant to chemo
Replenish blood cells help induce post-chemo recovery
with HSC growth factors
Host Toxicities anti-cancer Tx has a variety of common host toxicities
o Myelosuppression loss of WBC, platelets, & RBCs in trying to attack
blood cell tumor
o GI Toxicity methotrexate: nausea, vomiting, diarrhea, stomach
ulcers
o Alopecia hair loss
o Nephrotoxicity cis-platinum
o Cardiotoxicity anthracyclines (-rubicin): irreversible cardiomyopathy
o Neurotoxicity oxaliplatin (sensory
neuropathy), vinca alkaloids (peripheral neuropathy)
o Hemorrhagic Cystitis Ifosfamide accumulates in bladder, causes
lesions bleeding
o Bilary sclerosis irinotecan (not sure actually, but bile excretion after
UGT SN-38)
Tumor-Specific Delivery

Antibody-Mediated Delivery toxins/radioisotopes conjugated to

antibodies against tumor (CD20)
Regional Drug Delivery tumors have greater affinities for different regions
of body:
o Tumor-selective blood supply liver, brain, head/neck inject drug
directly here
o Regional advantage if drug in higher concentrations of tumor
region
Survival Pathway Inhibition block biochemical pathways which tumor
uses to survive & proliferate
o EGFR epidermal growth factor receptor, upregulated in many
tumors block this!

Exploiting Cell Cycle Checkpoint Deficiencies cancer cells have

uninhibited cell cycle checkpoints
o ATM/ATR Apical protein kinases which signal cell replication if
everything is A-OK
Normally if DNA damage occurs, ATM/ATR tells cell to halt
replication until fixed
Cancer this checkpoint is lost, replication continues despite
DNA damage
Anti-Cancer Therapy Cell Responses

Apoptosis best Tx response cancer cell will rapidly lyse

Mitotic Catastrophe next best excessive chromosomal disruption in
tumor cell, loss of proliferation
Senescence-Like prolonged cell cycle arrest without gross DNA damage;
proliferation stops temp/perm
Resistance tumor becomes resistant, either by slow proliferation, or
developing other metabolic means
o Be skeptical if drug company shows cells with inhibited proliferation
for only a few days, doesnt necessarily mean that this anti-cancer
drug actually has any significant clinical outcome
Anti-Cancer Therapy In Vivo Experimentation

Transplantable include xenografts & allografts

o Xenograft transfer a tumor from an immunologically different host
(human tumor lab rat)
Most common easiest, common because interested in
treating humor tumors
o Allograft transfer a tumor from an immunologically similar host (lab
rat tumor another rat)
Immunodeficient mouse induce an immunodeficiency in
mouse to cause tumor, can then transfer to immunocompetent
mouse & assess responses
o Ectopic/Orthotopic grafts can be placed in easy spot, or at spot of
transplant tissue origin
Ectotopic easier, but maybe not as accurate
Orthotopic more difficult, but will more accurately similar
tumor environment
Spontaneous induce a transgenic oncogene in lab rat to cause real
disease attempt to treat this
Maximum Tolerated Dose much higher in mice, one shortcoming
Dose Limiting Toxicity what toxicity occurs first, limiting the dose (e.g.
liver damage)
Anti-Cancer Therapy Clinical Trials

(Phase 0) see if drug does in fact work on its target in humans, rather than
just lab rats
Phase I identify sensitive organs, establish max
tolerated dose (escalation), patients w/ no options
Phase II apply doses & schedules of Phase I to a focused
population of target patients
Phase III compare new Tx vs. standard, conduct large scale RCTs, assess
for weird rare reactions

Cancer Cell Drug Resistance

Metabolic decreased prodrug activation, increased drug degradation

Uptake/Transport decreased drug uptake, increased drug efflux
Altered Target target molecule undergoes change/mutation to be resistant
to drug
Increased Target increased abundance of target, drug is outnumbered
Increased Repair repair processes in cancer cells upregulated, fix DNA
damage
Drug Resistance Development

Classical change in cell confers a specific resistance to a single drug

Multiple Drug Resistance (MDR) change in cell brings about resistance to
many drugs
o P-glycoprotein pumps drugs out, can confer a non-specific
resistance to many lipophilic drugs
o Wide variety of other MRPs many MDR proteins on various
chromosomes, not small problem
Log Cell Growth & Log Cell Kill

Drug

Mouse Experiment inject different amounts of cancer cell into mice, see
how long it took to be fatal
o Log Cell Growth because cells grow on a logarithmic scale, each log
factor increase of tumor would equate to a linear decrease in survival
time.
o Introducing chemotherapy chemotherapy was shown to extend
mouse life by the same amount, no matter what level of tumor burden
was present.
o Log Cell Kill because of above observation, realized that
chemotherapy kills the same % of tumor cells each time, not the same
number
Formal Log Cell Kill 1 log cell kill = 90% cells killed (reduce by factor of
10); 2 log = 99%, 3 = 99.9%
Treatment Resistance after one round of log cell killing, next
round wouldnt be as effective (resistant)
o Goldie-Coldman Hypothesis once a tumor gets large enough,
chance of MDR high
o Alternating Drug Schedules give different drug for each round of
log cell kill, avoid resistance
Gompertzian Growth theory that tumor cells actually grow faster at
lower numbers
Norton-Simon Hypothesis tumor killing is also proportional to growth
rate
o Outcome Tx of tumor in early phases will extend life the same
amount as Tx in late phases
o Early treatment only beneficial if Tx wipes out
tumor entirely need to be aggressive early!
o Sequential Therapy proved that AAABBB Tx better than
ABABAB Tx for tumors
Combination Therapy
Combination Therapy Principles:

Effectiveness each drug by itself should have some effectiveness in

treating tumor
o Diversity drugs used in combination should act thru
different mechs (less resistance chance)
o Non-overlapping Toxicity drugs should not have the same
toxicities bad SE!
Adjuvant Therapy therapy given after main treatment, to eliminate any
residual disease
o Tumor Example surgery followed by chemotherapy, to eliminate
hidden tumor cells
o Benefits can help cure patients who were not completely treated by
initial therapy
o Risks can subject cured patients to unnecessary injury must weigh
this against benefit
Neoadjuvant Therapy therapy given before main treatment, to reduce
chance of residual disease
o Tumor Example chemo before surgery shrink tumor to size that
can be surgically removed
Radiosensitization chemotherapy to make tumor cells more sensitive to
radiation
Damaging Agents

DNA

Alkylators include mechlorethamine & cyclophophamide, act to form

crosslinks with DNA:
o Mechlorethamine nitrogen mustard, highly reactive, give IV
o Cyclophosphamide prodrug activated into a mustard, give orally
Unusual toxicity can cause hemorrhagic cystitis give
early in day to avoid urine
accumulation, give lots of fuids to avoid
concentration

Ifosfamide similar to cyclophosphamide, but can

give MESNA to neutralize urine
o Mechanism drugs will crosslink with DNA to inhibit function
o Resistance Mech increased DNA repair, alkylator scavengers,
and decreased uptake
o Dose-limiting Toxicity usually bone marrow suppression, 23 wks to reach low point
Non-Alkylators include cis-platinum
o Cis-platinum although not true alkylating agent, has many similar
properties
Unusual toxicity severe nausea & vomiting, ototoxicity
o Mechanism drugs will crosslink with DNA to inhibit function
o Dose-limiting Toxicity causes renal toxicity (& renal excretion)
Carboplatin dose-limiting toxicity is myelosuppression (bone marrow
suppression)
Oxaliplatin dose-limiting toxicity is sensory neuropathy, has lifetime
cumulative dose limit
Antimetabolites

Methotrexate is a folate analog, binds tightly

to dihydrofolate reductase

Mechanism binding dihydrofolate reductase prevents folate

reduction (recycled), and thus
prevents dUMP dTMP methylation; DNA damage

Unusual Toxicity can cause GI mucositis

Dose-Limiting Toxicity usually myelosuppression, or GI mucositis
Drug-drug interaction be careful when giving
w/ neprhotoxic agents (renal excretion)
Fluorouracil 5-FU becomes ribosylated & altered to FdUMP,
inhibits thymidylate synthase
o Mechanism FdUMP tries to be converted to FdTMP by thymidylate
synthase, but F stops,
forms an irreversible complex

o
o
o

Leucovorin given with 5-FU, acts as a reduced folate cofactor for

thymidylate synthase,
increases formation of FdUMP-thymidylate synthase
complex

Resistance decreased DNA synthesis, increased thymidylate

synthase
o Metabolism 5-FU is rapidly metabolized, needs to be protected in
prodrug form to be effective
o Toxicity various depending on administration, has intra-individual
variations in metabolism
Dihydroxypyrimidine dehydrogenase (DPD) metabolizes 5-FU,
various levels in pts.
Cytosine Arabinsoide (Ara-C) cytosine analog, converted Ara-CTP put
into DNA, stops elongation
o Dose-Limiting Toxicity is myelosuppression
Gemcitabine cytosine analog, converted dFd-CTP put into DNA, stops
elongation
o Radiation Sensitizer will make GI tumor cells more susceptible to
radiation
o
DoseLimiting Toxicity is myelosuppression, also causes flu-like
symptoms
SKIPPED: Thiopurines guanine analog, converted to thioguanine put
into DNA
o Intra-individual variations TMPT metabolizes thiopurines
o Dose-Limiting Toxicity is myelosuppression
o Hyperuricemia from tumor lysis syndrome
Allopurinol prophylaxis against hyperuricemia
Chromosome Structure/Function Inhibitors

Microtubule Antagonisists include vinca alkaloids and taxanes

o Vinca alkaloids include Vincristine,
Vinblastine, Vinorelbin

Mechanism bind free tubulin dimers form

aggregates, cant form microtubules
Toxicity have a peripheral neuropathy,
also myelosuppression

 Resistance MDR
Taxanes include Paclitaxel, Taxotere
Mechanism bind to
assembled microtubles microtubules cant depolymerize
Toxicity induce a neutropenia
Topoisomerase II Inhibitors include anthracyclines (-rubicins)
o Anthracyclines include any -rubicin
Mechanism inhibit topoisomerase II (breaks both
strands) contorted DNA
Toxicity limited by myelosuppression and cardiac
arrhythmias
Unusual toxicity irreversible cardiomyopathy need
lifetime dose limit
Topoisomerase I Inhibitors include
o Camptothecins include Topetecan, Irinotecan
Mechanism inhibit topoisomerase I (breaks one
strand) contorted DNA
Toxicity can cause myelosuppression
Excretion different:
Topetecan excreted renal
Irinotecan a prodrug converted to active form (SN38), bile excretion
SKIPPED: Intra-Individual Variability
o

Morphometric variability based on body size & composition

Pathophysiological based on renal/hepatic function
Demographic based on age/gender
Genetics based on drug metabolism & transporter function
o Thiopurine S-methyltransferase (TPMT)
metabolizes thiopurines used to screw up DNA
o UDP-Glucuronosyltransferase (UGT) metabolizes ironotecan to
SN-38 bile excretion
o Dihydropyrimidine Dehydrogenase (DPD) metabolizes 5-FU
Overcoming Variability Problems accomplished through a number of
ways
o Phenotyping assess for phenotypes which have low/high
metabolism
o Genotyping assess for genotypes

Desai, A. and Mathis, M., Hematology, spring 2009. Open Michigan, Regents of the
University of
Michigan,http://open.umich.edu/education/med/m2/hematology/winter2009/materials
; (Accessed 23 December 2015). License: Creative Commons Attribution
(http://creativecommons.org/licenses/by/3.0/ ).