INTRODUCTION TO HEMOSTASIS

Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

OUTLINE KEY COMPONENTS OF HEMOSTASIS

I Introduction: Hemostasis
A What is Happening Outside & Inside of Blood Vessel CELLULAR COMPONENTS
II Key Components of Hemostasis • Platelets (Thrombocytes)
A Cellular Components
B Plasma Components • Cells of Vascular Intima: inner most lining of blood vessel.
III Phases of Hemostasis o Endothelial Cells: cells that line the vascular intima.
A Primary Hemostasis • Extravascular Tissue Factor Bearing Cells: coagulation
i Process for Primary Hemostasis factors.
ii Disease Affecting Primary Hemostasis o Fibroblasts
B Secondary Hemostasis o Smooth Muscle Cells
i Process for Secondary Hemostasis o Endothelial Cells
C Fibrinolysis
i Process for Fibrinolysis PLASMA COMPONENTS
• Coagulation Proteins (Clotting Proteins/Clotting Factors):
allows to form clots.
INTRODUCTION: HEMOSTASIS • Fibrinolytic Proteins: served as a balancer that helped to
dissolve the clot.
Complex Physiologic Process of Hemostasis • Inhibitors: acts as regulators only for needed process.
• Maintains circulating blood in a fluid state Table 1.1 Vascular Intima of the Blood Vessel
• Produces a clot to stop bleeding Innermost Vascular Lining
• Confines the clot to the site of injury • Endothelial Cells (Endothelium)
• Dissolves the clot during wound healing Supporting the Endothelial Cells
• Internal elastic lamina composed of elastin and
Thrombotic collagen
Disorder Cannot maintain fluid state. Subendothelial Connective Tissue
(Thrombosis)
• Collagen, and fibroblasts in veins
Hemorrhagic
Disorder Leads to uncontrolled bleeding. • Collagen, fibroblasts, and smooth muscles in arteries
(Hemorrhage)
PHASES OF HEMOSTASIS
Fibrinolytic • Primary Hemostasis (Clotting)
Unready to clot that leads to
Disorder • Secondary Hemostasis (Clotting)
another bleeding.
(Fibrosis) • Fibrinolysis (Dissolution of Clots)
• How Does Blood Knows When to Clot? PRIMARY HEMOSTASIS
o The inner lining of the blood vessels has the anticoagulant • First response to vascular injury.
properties. The blood draws out, it will go out, & clot • Activated by small injuries to blood vessels.
occurs. • Rapid and short-lived response.
o Known as the short-lived response due to the
WHAT IS HAPPENING OUTSIDE & INSIDE OF BLOOD replacement or enhancement by secondary
Outside the Inside the hemostasis for more clot (fibrin clot).
Blood Vessel Blood Vessel
Coagulation No Coagulation Components (Initiated/Facilitated by Cellular
(Hemostasis) Blood (Remains Fluid) Components) “Key Players”
• Vascular System (Arteries, Veins, Capillaries)
Abnormal Mechanism Abnormal Mechanism • Platelets
No Coagulation Coagulation
(Hemorrhagic Disease) (Thrombosis Disease) Process For Primary Hemostasis
1. Vasoconstriction: it is the first response to injury which
Blood Outside the Blood Vessel narrows the gap.
a. The vascular system contracts to seal the wound or
Normal Mechanism Coagulation (Hemostasis) reduce blood flow.
No Coagulation 2. Platelet Activation/Adherence/Secretion
Abnormal Mechanism a. Activation: platelets will be activated by tissue
(Hemorrhagic Disease)
factors and collagen.
b. Adherence: platelets will stick to blood vessels via
Blood Inside the Blood Vessel collagen; hence platelets adhere to exposed collagen.
No Coagulation (remains c. Secretion: platelets secrete granules to attract the
Normal Mechanism other platelets and initiate the aggregation.
fluid) 3. Platelet Aggregation: allows the platelet plug form from
Abnormal Mechanism Coagulation (Thrombosis) compiled or bind platelets.
4. Platelet Plug (Unstable): End-product
 INTRODUCTION TO HEMOSTASIS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

Disease Affecting Primary Hemostasis

Collagen • Poor platelet response.

Abnormalities • *walang kakapitan*
Thrombocytopenia • Decreased platelet count.
(Quantitative) • *wala yung pangkapit*
• The platelet quantity is
sufficient but can’t function
properly due to poor platelet
quality.
Qualitative Platelet
• Have problem with receptors,
Disorder
hence there will be a problem
on binding or sticking of
platelets to blood.
• *Wala yung pangkapit*
• Absence of binding site for
platelets.
Von Willebrand
• Von Willebrand (Bridge): a
Disease
factor that bind platelets to
collagen.

SECONDARY HEMOSTASIS
• Activation of coagulation proteins to form a fibrin clot. Factor III → Factor VIII → Factor X → Complex Fibrinogen
• Activated by large injuries to blood vessels. → (Thrombin) → Fibrin → Fibrin Clot
• Delayed, long-term response.
• Facilitated by circulating proteins or coagulation proteins Prothrombin (thrombin precursor) → complex X & V →
for the coagulation factors & inhibitors. Thrombin
• Begins immediately after primary hemostasis.
Table1.2 Primary & Secondary Hemostasis
Fibrin (Stable) End-product
Primary Hemostasis Secondary Hemostasis
Associated Coagulation Factor Deficiencies
Disease (leads to a hemorrhagic disorder) Activated by Activated by large injuries
Tube Used (For desquamation and small to blood vessels and
Orange (Rapid Serum) injuries to blood vessels surrounding tissues
Faster Clotting)
Precursor of thrombin due to Involves vascular intima Involves platelets and
Prothrombin Factor XV converting inactive and platelets coagulation system
thrombin to its active form Rapid, short-lived Delayed, long-term
An enzyme that converts response response
Thrombin fibrinogen to a localized fibrin Procoagulant substances
The activator, tissue
clot exposed or released by
factor, is exposed on cell
damaged or activated
membranes
Process For Secondary Hemostasis endothelial cells
1. Activation of zymogens (proenzymes).
2. Formation of complex that activates other zymogens. FIBRINOLYSIS
3. Generation of thrombin. • Final stage of coagulation.
4. Activation of fibrinogen to fibrin. • Begins a few hours after fibrin clot formation.
5. Fibrin stabilization by Factor XIII. • Systematic, accelerating hydrolysis of fibrin by bound plasmin.
6. Fibrin Clot (Stable): end-product o Activated by Fibrinolytic Proteins called Tissue
Plasminogen Activator (TPA), and Urokinase
Plasminogen Activator (UPA).
• The digestion of fibrin clot keeps the vascular system free
of deposited fibrin or fibrin clot.
• The fibrin clot can clog the blood vessels if present
permanently.
TPA & UPA Converts plasminogen to plasmin
Dissolves or hydrolyzes fibrin clot
Plasmin
into its fibrin degradation product
 INTRODUCTION TO HEMOSTASIS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

Inhibitors of Fibrinolysis
• Inhibits TPA.
Plasminogen Activator
• Inhibits activator.
Inhibitor-1 (PAI-1)
• Serves as control.
Alpha 2 - Antiplasmin • Inhibits plasmin.
(a2-AP) • Inhibits activation.
Thrombin-Activable • Removes fibrin binding
Fibrinolysis Inhibitor sites.
(TAFI) • Inhibits binding site.

Process For Fibrinolysis

1. Incorporation of plasminogen, TPA, UPA, and PAI-1 to fibrin
clot.
2. Conversion of plasminogen to plasmin by TPA and UPA.
3. Fibrin digestion of plasmin.
4. Fibrin Degradation Product: end-product of fibrinolysis.

Plasminogen → (TPA, UPA) → Plasmin (hydrolysis of fibrin

clot)

Fibrinogen → (Thrombin) → Fibrin Clot (clot formation)

 TOPIC TITLE LOREM IPSUM
Ms. A.N. Sarmiento| 2nd Semester | A.Y. 2023-2024

OUTLINE HEMATOPOEITIC STEM CELL

1. Megakaryocytopoiesis
• Origin of blood cells
• Platelets
• Megakayocytes • Capable of self-renewal
• Hematopoietic Stem cells - Never run out of supply of hematopoietic stem cell
• Thrombopoietin • Pluripotent
2. Megakaryocytes differentiation and progenitors - One stem cell that can differentiate into different
3. 3 Stages of megakaryocyte cell types
4. Endomitosis • Can undergo self-renewal, differentiation, or
• Stages apoptosis (programmed cell death)
• Methods of Identification
5. Morphological identifiable stages
Thrombopoietin
• Terminal Megakaryocytes Differentiation Stages
6. Notifiable Characteristics - Major growth factor for megakaryocytopoiesis
• MK I – Megakaryoblast - A hormone found in the liver, kidney, bone marrow
• MK II – Promegakaryocyte and smooth muscles
• MK III – Megakaryocyte
7. Thrombocytopoiesis (Platelet Shedding) MEGAKARYOCYTE DIFFERENTIATION AND
8. Hormones And Cytokines Of Megakaryopoiesis PROGENITORS
• Thrombopoietin
• Recombinant TPO
• Cell Derived Stimulators of
Megakaryocytopoiesis
9. Summary

MEGAKARYOCYTOPOIESIS
• A process of the production and maturation of
thrombocytes or platelets
• Occurs in the bone marrow

PLATELETS
• Also known as Thrombocytes
• Non-Nucleated blood cells • Megakaryocyte localize near the endothelial cell lining
- Because they are only cytoplasmic fragments of of venous sinus of the bone marrow
Megakaryocyte
• Venous sinus - pathway to the peripheral blood
• Size: 2.5 um, MPV (8 to 10 fL) circulation
• Reference Range: • It is important so that if it releases platelet, the platelets
- N.V. 150-400 X109/ L will go directly to the circulation
- Higher in women
- Slightly lower at both sexes over 65 yrs. Old
3 STAGES OF MEGAKARYOCYTE
• 30% (1⁄3) of platelets are in the spleen or also known
as the resting platelets
• 60% (2⁄3) can be found in the circulation or also known
as the circulating platelets
• # PLT/OIF in PBS (using Wright stain): 7-21 platelets
• Function: Trigger Primary Hemostasis
• Arise from megakaryocytes

MEGAKARYOCYTES
• Largest cell in the bone marrow
• Less than 0.5% of all bone marrow cells
- 1 megakaryocyte is equivalent to 2,000 platelets
• With multiple chromosome copies (polyploid)
- Can reach up to 128 polyploid due to endomitosis
• In the bone marrow, megakaryocytes should NOT be
count in a peripheral blood
• Bone marrow aspirate (Wright stained)
- Size: 30-50 um 1. Proliferative Phase
- Nucleus: multilobulated 2. Terminal differentiation
• 2-4 megakaryocytes per 10x low power field 3. Platelet Shedding
 TOPIC TITLE LOREM IPSUM
Ms. A.N. Sarmiento| 2nd Semester | A.Y. 2023-2024

ENDOMITOSIS
• Where a cell continues DNA replication and
cytoplasmic maturation but does not divide
• It is a form of mitosis that lacks telophase and
cytokinesis.
• It is run by through transcription factors called GATA-1
and FOG1
- GATA-1 and FOG1 -> RUNX1 -> ENDOMITOSIS
➢ GATA-1 - Globin Transcription Factor 1
➢ FOG1 - Friend of GATA-1
• Endomitosis is important because even if there is no
cell division, the multiple DNA copies in the cell will
allow megakaryocytes to build a lot of cytoplasm
- Abundant cytoplasm = abundant platelets
TERMINAL MEGAKARYOCYTE DIFFERENTIATION STAGES
STAGES
1. BFU- Meg
- Burst forming unit- Megakaryocyte
- Least Mature Progenitor Cell
2. CFU-Meg
- Colony forming unit- Megakaryocyte Diploid
- Participate in Normal Mitosis
3. LD-CFU-Meg
- Low density – colony forming unit –
Megakaryocytes
- Most Mature Progenitor Cell
- No mitosis/Do not have the capacity to divide
(endomitosis)

METHODS OF IDENTIFICATION
1. Immunologic Tests (Flow Cytometry)
2. Cytochemical Stains

• Increase in size as it matures

• Nucleus becomes more lobulated
• Chromatin (more condensed) and nuclei less visible
• NC ratio decreases because cytoplasm content
increases through endometriosis

NOTIFIABLE CHARACTERISTICS
MK I - Megakaryoblast
• When looking at the bone marrow aspirate, these three • Plasma blebs
look the same. ➢ Blunt projections from the margin that
• All 3 resemble lymphocytes in bone marrow Wright resemble platelets.
stained smear. • Beginning of the development of demarcation system
• All 3 cannot be differentiated morphologically. ➢ DEMARCATION SYSTEM - channels present
• Each progenitor cell can be identified through their in the cell that allow it to have indentation in
markers plasma (they grow inward)
MK II - Promegakaryocyte
MORPHOLOGICAL IDENTIFIABLE STAGES • Slightly lobulated nucleus
(Terminal Differentiation) MK III - Megakaryocyte
• MK I – Megakaryoblast • Intensely lobulated nucleus
• MK II – Promegakaryocyte
• MK III – Megakaryocyte
 TOPIC TITLE LOREM IPSUM
Ms. A.N. Sarmiento| 2nd Semester | A.Y. 2023-2024

RECOMBINANT TPO
• Used as a medication for individuals who cannot
produce or low in TPO leading to thrombocytopenia
2. Cell Derived Stimulators of Megakaryocytopoiesis
a. IL3
• For early differentiation of stem cells
a. IL6 and 11
• Act in the presence of TPO to enhance endometriosis,
megakaryocyte maturation and platelet release

• IL - Interleukin
• TPO & IL-11 can be used as Therapeutic Agent for
thrombopoietic people

SUMMARY

THROMBOCYTOPOIESIS (PLATELET SHEDDING)

• Situated near the border venus sinus
• In Thrombocytopoiesis, the cytoplasm of the
megakaryocytes extends across the lining of the
sinusoidal to release cytoplasmic fragments
• 1 MK-III may produce 2000-4000 platelets
• Mechanism of release:
1. Demarcation (invagination)
2. Fragmentation of the cytoplasm
3. Platelets are formed at the ends of proplatelets
and released by microtubular action.
• Thrombocytopoiesis leaves behind naked
megakaryocyte nuclei to be consumed by bone
marrow macrophages

HORMONES AND CYTOKINES OF MEGAKARYOPOIESIS

1. Thrombopoietin
• Most important
• Induce stem cell differentiation
• Induce proliferation and platelet release
• Found in kidney, LIVER (majority), smooth muscles,
and bone marrow
• Circulates in the plasma
 VASCULAR INTIMA IN HEMOSTASIS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

OUTLINE • Arteries need thick layer because it is where the pressure

is present when injecting blood from heart to the different
I Introduction
A Blood Vessels (Vasculature) tissue.
B Endothelial Cells
II Anticoagulant Properties of Intact Vascular Intima • Once a blood vessel is damaged,
A The Structure of Blood Vessel Wall Collagen collagen is exposed to platelets.
i Rhomboid Endothelial Cell • Activated platelets = Platelet clot
ii Prostacyclin (Prostaglandin I2)
iii Nitric Oxide (NO) Fibroblasts Allows to produce a collagen
iv Tissue Factor Pathway Inhibitor (TFPI)
v Thrombomodulin Smooth
vi Heparan Sulfate Muscle Used for the constriction of artery
vii Tissue Plasminogen Activator (TPA) Cells
III Procoagulant Properties of Damaged Vascular Intima
IV Fibrinolytic Properties of Vascular Intima ENDOTHELIAL CELLS
• Complex and heterogenous cells.
INTRODUCTION • It displays a unique structural and functional
characteristic depending on the environment and tissue
BLOOD VESSELS (VASCULATURE) location.
• Vessels that carry blood throughout the body. • In the blood vessels, ECs form a smooth, unbroken
surface that eases the fluid passage of blood.
• Provides the interface between circulating blood and the
body tissues. • Supported by a basement membrane and connective
tissue layer.
3 Layers of Blood Vessels • It is highly involved in hemostasis through their
anticoagulant, procoagulant, and fibrinolytic properties.
• Vascular Intima. ❖ Take Note! Hemostasis is not only about clot formation, but
• Is the layer that has the direct also a complex physiological process that maintains
contact in the circulating blood. balance.
Inner
Layer • This innermost lining of blood
vessels is a monolayer of
metabolically active endothelial
cells (ECs).
Lined By Monolayer of Active Endothelial Cells
Middle
Vascular Media
Layer
Outer
Vascular Adventitia
Layer

Table 3.1 Vascular Intima of the Blood Vessel

Innermost Vascular Lining
• Endothelial Cells (Endothelium)
Supporting the Endothelial Cells
ANTICOAGULANT PROPERTIES OF INTACT VASCULAR
• Internal elastic lamina composed of elastin and INTIMA
collagen • Anticoagulant Properties of Intact Vascular Intima.
Subendothelial Connective Tissue • Normally the intact vascular endothelium prevents
thrombosis by:
• Collagen, and fibroblasts in veins o Inhibiting platelet aggregation
• Collagen, fibroblasts, and smooth muscles in o Preventing coagulation activation and propagation
arteries o Enhancing fibrinolysis

THE STRUCTURE OF BLOOD VESSEL WALL

Rhomboid Endothelial Cell

• Presents a smooth, contiguous surface.
• The smooth surface prevents any obstruction of the cells.
• The contiguous feature makes EC a shield or physical
barrier between platelets circulating & procoagulant
substances present underneath this layer. It promotes
platelet adhesion, and Tissue Factor in Fibroblasts and
smooth muscle cells that activates coagulation.
 VASCULAR INTIMA IN HEMOSTASIS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

Table 3.2 Anticoagulant Properties of Intact Vascular

Endothelium
Endothelial Cell
Anticoagulant Property
Structure/Substance
Vascular endothelium is
Present a smooth,
composed of rhomboid
contiguous surface
cells
The eicosanoid platelet
ECs secrete prostacyclin
inhibitor
ECs secrete nitric oxide A vascular “relaxing” factor
ECs secrete the An anticoagulant that
glycosaminoglycan regulates thrombin
Prostacyclin (Prostaglandin I2) heparan sulfate generation
• A platelet inhibitor and vasodilator prevent unnecessary A regulator of the extrinsic
platelet activation. ECs secrete TFPI
pathway of coagulation
• Synthesized through the Eicosanoid pathway.
An integral component of
Nitric Oxide (NO) ECs express the protein C
the protein C control
• Vascular Relaxing Factor that counteracts receptor EPCR
system
vasoconstriction.
• It inhibits platelet activation and promotes angiogenesis ECs express cell
A protein C coagulation
and healthy arterioles. membrane
control system activator
• Secreted by smooth muscles, neutrophils, and thrombomodulin
macrophages. ECs secrete TPA Activates fibrinolysis
Tissue Factor Pathway Inhibitor (TFPI)
• It limits the activation of the TF:VIIa; Xa complex. PROCOAGULANT PROPERTIES OF DAMAGED VASCULAR
• Inhibits Extrinsic Coagulation Pathway. INTIMA
• Smooth muscle cells induce vasoconstriction, and mostly
Thrombomodulin present in arteries.
• Activates the Protein C Pathway (digest Factors Va and • Collagen binds and activates platelets under subendothelial
FVIIIa) layer,
• Inhibits thrombin formation. • Endothelium Cells (EC) secrete vWF.
• Thrombin + Thrombomodulin = Activate PCP (No Fibrin Clot) o Served as bridge between platelet and collagen,
especially in area of high shear stress.
Heparan Sulfate o Described as a “carpet” on which activated platelets
• Enhances activity of antithrombin, and a glycosaminoglycan assemble.
and inhibits thrombin by enhancing antithrombin. o Ultra large, need to be cleaved by ADAMTS13 for it to
o Antithrombin: a blood plasma serine protease be into shorter chains that support normal platelet
inhibitor. adhesion.
• Retards coagulation by activating antithrombin, and o Artery needs vWF to bind collagen and platelet if vein
fibrinogen will not be converted to fibrin. platelet can directly adhere to collagen.
Tissue Plasminogen Activator (TPA) • The EC secrete adhesion molecule that promotes platelet
and leukocyte binding.
• Activates fibrinolytic system specifically plasmin.
o P-Selectin: an adhesion molecular that promotes
• Converts plasminogen to plasmin. platelet and leukocyte binding.
o ICAMS: Intercellular adhesion molecules
o PECAMS: Platelet endothelial cell adhesion molecules
• Smooth Muscle Cells and Fibroblast
o It exposes tissue factor that support the constitutive
membrane protein tissue factor.
• EC in inflammation
• Tissue factor is induced by inflammation.
• Inflamed individuals are prone to coagulation.
❖ Nice to Know! storage site of Von Willebond Factor is
Weibel - Palade Bodies.
2 Important Procagulant
• Thromboxane
• ADP
 VASCULAR INTIMA IN HEMOSTASIS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

Table 3.3 Procoagulant Properties of Damaged Vascular Intima

Structure/Substance Procoagulant Property
Smooth muscle cells in
Induce vasoconstriction
arterioles and arteries
Exposed subendothelial
ECs secrete prostacyclin
collagen
Important for platelet
binding to collagen at site
Damaged or activated of injury: platelet adhesion
ECs secrete VWF as a
first line of defense against
bleeding
Damaged or activated
Promote platelet and
ECs secrete adhesion
leukocyte binding and
molecules: P-selectin,
activation at site of injury
ICAMs, PECAMs
Exposed smooth muscle Tissue factor exposed on
cells and fibroblasts cell membranes
Tissue factor is induced by
ECs in inflammation
inflammation

FIBRINOLYTIC PROPERTIES OF VASCULAR INTIMA

• Tissue Plasminogen Activator (TPA)
o Activates fibrin bound plasminogen to form plasmin
(plasmin digest thrombus and restores blood flow)
• Plasminogen Activator Inhibitor 1 (PAI-1)
o Inhibits activation of fibrinolytic system
o Plasminogen to plasmin, TPA is needed, PAI-1 will
inhibit TPA.
o Avoids the digestion of fibrin clot.
• Thrombin activatable fibrinolysis inhibitor (TAFI):
activated by the thrombin bound to EC membrane
thrombomodulin; inhibits the Fibrinolysis.
• ADAMST - 13: works with Von Willebrand factor.
 Chapter #4: Mechanism of Primary Hemostasis
Ms. Aila Nell Sarmiento | 2nd Semester | A.Y. 2023-2024

OUTLINE
• Platelet Function AGGREGATION
o Adhesion • Platelet- platelet adherence
I. What happens during platelet • Irreversible
adhesion? • Platelet Plug formation
o Aggregation Requires:
I. What happens during platelet • Fibrinogen (Factor I) and Ionized Calcium
aggregation? • GP IIb/IIIa receptor
II. White Clot Associated diseases:
III. Red Clot • Afibrinogenemia
o Secretion ✓ Absence of fibrinogen
I. Substances Secreted by Platelets • Hypofibrinogenemia
• Primary Hemostasis Laboratory Tests ✓ Deficiency of fibrinogen
A. Platelet Count • Glanzmann Thrombasthenia
B. Reasons Why Platelets are Hard to Count ✓ Absence of GP IIb/IIIa receptor
C. Three Methods of Platelet Count What Happens During Platelet Aggregation?
✓ Platelet Estimation in PBS • Generation of “collagen and thrombin activated
D. Peripheral Film Preparation (COAT)” platelet (PLT) integral to cell based
E. Peripheral Blood Smear coagulation model
✓ Well-Made Peripheral Blood • TXA2 and ADP trigger “inside out activation” of GP
Smear IIb/IIIa (αIIbꞵ3) to bind fibrinogen and vWF
F. Estimation of Platelet Count
• P-selectin (CD 62) supports adhesion of platelets with
✓ Performing a Platelet Estimate leukocytes
✓ Reporting of Platelet Estimation
• Platelet shape change from discoid to round and
Result
extend pseudopods
G. Manual Platelet Count
During aggregation, two clots are formed:
✓ Direct Method
1. White clot
✓ Indirect Method
2. Red clot
H. Automated Platelet Count

PLATELET FUNCTION
ADHESION
• Platelets cling to non-platelet surfaces (collagen)
• Secrete growth factors
• Reversible
Requires:
➢ vWF (von Willebrand Factor)
✓ Serves as the bridge connecting the
platelets and collagen in arteries
✓ Produced in Endothelial Cell (Weibel-Palade
Bodies) and Platelet (Alpha Granule)

➢ GP Ib/IX/V receptor
Associated diseases:
• Bernard-Soulier syndrome
✓ Absent GP Ib/IX/V
✓ This will not allow platelet adhesion
• vWF disease
✓ Missing/defective vWF WHITE CLOT
• Platelet + vWF
What Happens During Platelet Adhesion? • Arteries and arterioles (AA -White)
1. GP Ib/ix/V binds vWF • Abnormal mechanism: AMI, Stroke, Peripheral Artery
2. GP VI binds collagen Disease
❖ additional binding of collagen RED CLOT
3. Bound GP VI initiates the release of thromboxane A2 • Platelet + vWF + Fibrin + RBC
(TXA2) and adenosine diphosphate (ADP) which • Veins and Venules (VV -Red)
activate α2ꞵ1 & αIIbꞵ3 • Abnormal mechanism: Venous Thromboembolic
✓ α2ꞵ1: Enhances or stabilizes platelet Disease
adhesion SECRETION
✓ αIIbꞵ3: Prepares or supports for aggregation • Activated platelets release granular contents
✓ Granules: Alpha Granules and Dense
Granules
• Irreversible
 Chapter #4: Mechanism of Primary Hemostasis
Ms. Aila Nell Sarmiento | 2nd Semester | A.Y. 2023-2024

• Occurs during aggregation

• Platelets secretes substances to further enhances PRIMARY HEMOSTASIS LABORATORY TESTS
the primary hemostasis A. Platelet Count
What Happens During Platelet Secretion? • Reference Range NV: 150,000 - 400,000/uL
1. Alpha granules & lysosomes content flow through • 150- 400 x 10^9/L
SCCS Thrombocytosis:
2. Dense granules contents are secreted through the ✓ Above 400, 000/ uL
plasma membrane ✓ Seen in Polycythemia Vera, idiopathic
Substances Secreted By Platelets thrombocythemia, CML, splenectomy
✓ Splenectomy: 30% of the platelets are
found in the spleen

Thrombocytopenia:
✓ Decrease Platelet Count
✓ Seen in Thrombocytopenia purpura, aplastic anemia,
acute leukemia and following chemotherapy/radiation
B. Reasons Why Platelets are Hard to Count
• Platelet adhere on foreign surfaces such as wall on
the pipettes
• Platelet easily disintegrates
• Hard to differentiate from debris because of the size
• Unevenly distributed in the blood
✓ Platelets tend to clump together
METHODS OF PLATELET COUNT
1. Platelet Estimation
2. Platelet Count (Direct, Indirect)
Platelet Estimation In PBS
Peripheral Blood Smear (PBS) are made from:
• Fresh drop of capillary blood without anticoagulation
• Fresh venous sample collected in EDTA
Within 2-3 hours of collection: Rodak 5th edition
Within 1 hour of collection: Mc Call
❖ Blood films from EDTA that remain at room temperature
for more than 5 hours produce artifacts.
WELL MADE PERIPHERAL BLOOD SMEAR

• It should covered ⅔ to ¾ of the glass slide

• Thick at the frosted end and becomes progressively
thinner toward the opposite end
• It should have feathery edge
• Zone of Morphology: area of optimal thickness for
light microscopic examination
✓ Should be at least 2cm in length
✓ Area to observe morphological structures
and perform Platelet Estimate
• Smear should occupy the central area of the slide and
be margin-free at the edges.
 Chapter #4: Mechanism of Primary Hemostasis
Ms. Aila Nell Sarmiento | 2nd Semester | A.Y. 2023-2024

ESTIMATION OF PLATELET COUNT

• Normal wedge smear demonstrates: 7-21 platelets
per field
• Counting the number of platelets seen in 10 OIF
Calculation:
• Average number of platelets/OIF x 20,000
• In instances of significant anemia or erythrocytosis,
use the formula below for the platelet estimate

Performing A Platelet Estimate

1. Select an area of the blood film in which most RBCs
are separated from one another with minimal
overlapping
✓ in Zone of Morphology LIGHT MICROSCOPY METHOD
2. Using the 100x OIO, count the number of platelets in Rees And Ecker’s
10 consecutive fields, and calculate the average • Diluting Fluid is composed of:
number of platelets per field ✓ Brilliant Cresyl Blue (stains the platelets)
3. To obtain the platelet estimate per uL of blood, multiply ✓ Sodium Citrate (prevents coagulation)
the average number of platelets by 20,000 ✓ Distilled Water (provides low specific gravity)
4. Compare the instrument platelet count with the platelet ✓ Preserves RBC which may be counted
estimate from the blood film simultaneously.
❖ PBS is done as a QC check for the automated result. ✓ Prevent adhesion of platelets
Procedure:
1. Rinse RBC pipet with Rees and Ecker DF
2. Aspirate blood up to the .5 mark of the RBC thoma
pipet
3. Aspirate the diluting fluid up to the 101 mark
Reporting of Platelet Estimation Result 4. Shake pipet (3-5 minutes)
5. Discard few drops

6.Charge the counting chamber. Stand for at least 3

minutes
7. Count Platelets: 4 corner large squares
Guy and Leake’s
• Diluting Fluid is composed of:
MANUAL PLATELET COUNT ✓ Crystal Violet
Direct Method ✓ Sodium Citrate
• Direct Method/Hemocytometer ✓ Distilled Water
• Whole blood is diluted with platelet diluting fluid in ✓ Formalin (40%)
a RBC pipet and counted in a hemocytometer Procedure:
➢ Light Microscopy Method 1. Aspirate blood up to the .5 mark of the RBC thoma
✓ Rees and Ecker’s pipet
✓ Guy and Leake’s 2. Aspirate the diluting fluid up to the 101 mark
➢ Phase Microscopy Method 3. Shake pipet (3-5 minutes)
✓ Recommended as it facilitate easier reading of 4. Discard few drops
platelets 5. Charge the counting chamber. Stand for atleast 3
✓ Brecker-Cronkite Method minutes
6. Count Platelets: 25 intermediate square of in central
large square
 Chapter #4: Mechanism of Primary Hemostasis
Ms. Aila Nell Sarmiento | 2nd Semester | A.Y. 2023-2024

PHASE MICROSCOPY METHOD

Brecker-Cronkite Method
• Procedure: Same as Rees and Ecker
• Diluting fluid used:
✓ 1% Ammonium oxalate
✓ This diluting fluid lyses RBCs and allows
platelets to form pseudopods (irregular
shapes)
• Microscope used:
✓ Phase Contrast Microscope
Indirect Method
• Platelets are counted in relation to 1,000 RBCs in
the blood smear.
• Not so reliable since results depend on the
distribution of the platelets and on the RBC count.
• Advantage: Allows study of platelet morphology
• Calculation:
✓ (RBC count / 1,000) x No. of PLT counted =
PLT count/cumm
• Dameshek’s
• Fonio’s
• Olef’s: the best indirect method but somewhat
hard to do
AUTOMATED PLATELET COUNT
• Anticoagulant used: EDTA
• Blood cells with the size of 2-20 fL is enumerated
as platelets
 TOPIC TITLE LOREM IPSUM
Mr./Ms. Professor | 2nd Semester | A.Y. 2023-2024

OUTLINE ✓ Hemoglobinopathy, same with microcytic RBC

• Causes of Falsely Low Platelets BLEEDING TIME
• Causes of Falsely Elevated Platelets • Original test for platelet function
• Bleeding Time • Screening test: Platelet Function and vWF
A. Duke’s Method • Focuses more on quality of primary hemostasis,
B. Simplate/ Surgicutt Method platelet function, and how well your body can form
C. Conditions Associated with Prolonged platelet plug
Bleeding Time • History:
D. Factors Affecting Bleeding Time o 1912: First described by Duke
• Correlation of Platelet Count and Bleeding Time o 1941: Modified by Ivy
• Platelet Adhesiveness Test o 1969: Mielke - standardization of bleeding
• Platelet Aggregometry time
A. Platelet aggregation test • Templated lancet: incision depth
✓ Whole Blood • Blood pressure cuff: 40 mmHg
✓ Platelet Rich Plasma o 1976: Surgicutt Bleeding Time Device
• Optical Aggregometry Tracing
• Platelet Lumiaggregometry A. Duke’s Method
A. Aggregating Agents Procedure:
• Clot Retraction Time 1. Cleanse the site
A. Hirschboeck (Castor Oil Method) 2. Make a skin puncture of not more than 2 mm depth
B. Stefanini Method 3. Start the timer immediately
C. Mac Farlane 4. Without touching the wound, blot the drop of blood with
• Capillary Fragility Test filter paper every 30 seconds
A. Rumple-Leede Test 5. Stop the timer as soon as the last drop of blood
disappears
CAUSES OF FALSELY LOW PLATELETS 6. Record: to the nearest 30 seconds — if the bleeding
1. Activation of platelets during venipuncture time goes beyond the normal range, the Phlebotomist
2. Partial clotting of specimens must wait for the timer to exceed 20 mins.
✓ Inversion is significant 7. Inspect the puncture site and provide post-puncture
3. Platelet satellitism care.
✓ Formation of platelet rosette • Normal values: 2 - 4 minutes
✓ 4 or more platelets on the cytoplasmic margin B. Simplate/Surgicutt Method
of a neutrophil or band Procedure:
✓ Effect: Pseudothrombocytopenia (false 1. Select a site on the volar area of the forearm
decrease in PLT count) 2. Place a sphygmomanometer cuff on the upper arm
✓ Remediation: Re-draw blood using Sodium 3. Prepare Simplate / Surgicutt (5mm wide, 1mm depth)
citrate Light Blue Top 4. Inflate sphygmomanometer cuff to 40 mmHg
✓ Correction for dilution = PLT count obtained 5. Depress the trigger and simultaneously start the timer
from light blue top x 1.1 6. Every 30 seconds, blot with filter paper until it stops.
4. Giant platelets • Normal values: 2 - 9 minutes
✓ If too big it will either be counted as rbc or C. Conditions Associated With Prolonged Bleeding Time
wbc ▪ Thrombocytopenia
5. Edta-induced platelet aggregation ▪ Functional platelet disorder
✓ Platelets form large clumps as large as WBC o vWD
✓ Effect: Pseudothrombocytopenia and o Glanzmann Thrombasthenia
Pseudoleukocytosis ▪ Vascular disorder
✓ Remediation: re-draw blood using Light Blue o Scurvy
Top o Vasculitis
✓ Correction for dilution = PLT count obtained ▪ Nonsteroidal Anti-inflammatory Drugs (NSAIDs)
from light blue top x 1.1 Therapy
✓ Correction for dilution = WBC count o Reduce inflammation and relieve fever and
obtained from light blue top x 1.1 pain by blocking enzymes and proteins
made by the body
CAUSES OF FALSELY ELEVATED PLATELETS o Aspirin: Avoid 7-10 days before bleeding
1. WBC fragments time procedure
2. Cryoglobulins ❖ does not block prostaglandin
✓ In patients with Cryoglobulinemia o Ibuprofen and Naproxen
(Mycoplasma Pneumonia Infection) ❖ Block prostaglandin (makes heavy
✓ Appear as small round inclusions/globules menstrual bleeding worse)
(intracellular & extracellular)
3. Microcytic RBC or red cell fragments falling below
upper threshold for platelet counts
4. Hemoglobin c
 TOPIC TITLE LOREM IPSUM
Mr./Ms. Professor | 2nd Semester | A.Y. 2023-2024

▪ Higher electrical impedance result means better

D. Factors Affecting Bleeding Time aggregation
1. Non platelet variables of intracapillary pressure
2. Skin thickness at the puncture site Platelet Rich Plasma (PRP)
3. Size and depth of the wound ▪ Instrument used: Light Transmittance Aggregometer
✓ Springloaded and c- activated lancet so the cut will o Photometer: distributed by Chrono Log or
always be standard Bio Data companies
CORRELATION OF PLATELET COUNT AND ▪ Original specimen: 9-12 mL of whole blood
BLEEDING TIME o Spin for 30 minutes at 50x g
1. Normal platelet count + prolonged BT o Stand for 30 minutes after centrifugation
o Qualitative platelet abnormality ▪ Platelet count of PRP: 200,000 - 300,000 / uL
o Primary vascular abnormality ▪ Stopper maintained: To maintain pH of sample
o von Willebrand’s syndrome ▪ Incubator 37 C for 5 mins
2. Low platelet count + normal BT ▪ Higher transmittance indicated better Aggregation
o Autoimmune thrombocytopenia
3. Low platelet count + very prolonged BT VII. OPTICAL AGGREGOMETRY TRACING
o Simultaneous quantitative and qualitative
platelet deficiency
PLATELET ADHESIVENESS TEST
▪ AKA Salzmann Test
▪ Used to determine: decrease vWF
▪ Platelet Count 1: Routine, collection
▪ Platelet Count 2: Collecting through glass bead
collection system
o Because platelets adhere to the glass, lower
platelet count is expected.
▪ Normal values: 26-60%
PLATELET AGGREGOMETRY
▪ For assessment of platelet function
▪ In vitro test to determine the ability of platelets to
aggregate with certain agonist
▪ Samples used: Whole Blood, Platelet rich plasma
▪ Highly complex test
▪ Can measure platelet adhesion, aggregation, and
secretion
▪ Result is measured with Electrical Impedance 5 Phases Of Platelet Aggregation
▪ Visualized using Aggregometry Tracing 1. Resting Platelet: stable baseline
▪ Not a routine test
2. Shape change: once agonist is added
o -Requested for patients who monitor
Antithrombotic Therapy 3. Primary aggregation
o For monitoring of platelet response to 4. Secretion
antiplatelet drugs 5. Secondary aggregation
❖ Aspirin - blood thinner; inhibit Cyclooxygenase (in DTS PLATELET LUMIAGGREGOMETRY
for Eicosanoid Pathway) ▪ Sample: WB or PRP
▪ Simultaneous measurement of platelet aggregation
A. Platelet Aggregation Test
▪ Anticoagulant used: Sodium Citrate
and secretion of ATP
Whole Blood Platelet Aggregometry ▪ Simplifies the diagnosis of platelet dysfunction
▪ Used in Whole Blood Platelet Aggregometry ▪ Principle: ATP oxidizes Luciferin-Luciferase reagent to
▪ Measured by electrical impedance generate cold chemiluminescence
o Same principle in CBC machine ▪ Thrombin
o There is an electrode with direct current
o Typically the first agonist used
o Changes in the impedance of the electrode
is measured = Result is graphed in Optical o Induces full secretion
Aggregometry Tracing A. Aggregating Agents
▪ Measured by electrical impedance ▪ Have certain aggregation patterns in Optical
▪ Diluted Specimen 1:1 (NSS) Aggregometry Tracing
▪ Holding temp.: 18-24C o Monophasic pattern: Either primary or
o Chilling = destroys platelet activity
o Centrifugation is not required, it can
secondary aggregation is present
activate the platelets o Biphasic pattern: Both primary or secondary
▪ Incubation: 37C for 5 mins aggregation is present
▪ Must be tested within 4 hrs of specimen collection
 TOPIC TITLE LOREM IPSUM
▪ Final concentration: 500 um
Mr./Ms. Professor | 2nd Semester | A.Y. 2023-2024

Aggregating Agents
1. Thrombin ▪ Aggregation Pattern
2. Synthetic Trap o Monophasic Pattern
3. ADP ❖ No lag phase
4. Epinephrine ❖ Rapid secondary aggregation
5. Collagen ▪ Assess viability of Eicosanoid Pathway
6. Arachidonic acid o Arachidonic Acid provides phospholipids in
7. Ristocetin Eicosanoid Pathway
8. ▪ Useful in detecting Aspirin related defect
5. Ristocetin
▪ Platelet membrane receptor: GPIb/IX/V
▪ Aggregation Pattern
o Monophasic Pattern
❖ Involves a little platelet shape
change and little secretion
▪ Final concentration: 1 mg/dL
▪ For checking deficiencies of vWF

1. Thrombin & TRAP (Synthetic Version Of Thrombin)

▪ Synthetic Form: Thrombin Receptor Activity Peptide
(TRAP)
▪ Biphasic Pattern
▪ Platelet membrane receptor: PAR1, PAR4, GP V, GP
Ibalpha
▪ Final concentration: 1 U/mL
▪ ATP secretion: 1.0-2.0 nm
▪ Induces full aggregation and secretion
2. Epinephrine
▪ Platelet membrane receptor: alpha 2 Adrenergic
Receptor
▪ Same pathway as ADP
▪ Final concentration: 2-10 ug/mL
▪ Cannot be used for whole blood testing Biphasic
Pattern
3. Collagen
▪ Platelet membrane receptor: GPIaIIa, GP VI
▪ Aggregation Pattern
▪ Monophasic Aggregation
o With lag phase: stimulates platelets to
release granule contents
o No primary aggregation
❖ Final concentration: 5ug/mL
4. Arachnidonic Acid
▪ Platelet membrane receptor: TPalpha, TPbeta
 TOPIC TITLE LOREM IPSUM
Mr./Ms. Professor | 2nd Semester | A.Y. 2023-2024

CLOT RETRACTION TIME C. Mac Farlane

▪ Directly proportional to platelet count Procedure:
▪ Degree of clot retraction: Measured on the amount of 1. Extract 5 mL of blood
expressed serum 2. Transfer into the test tube
▪ Normal clot retraction requires: 3. Place an applicator stick inside the tube and cover it
o Normal number of Platelets with a cork
o Calcium 4. Incubate for 1 hour (water bath). Observe for
o ATP coagulation every 5-10 minutes
o Fibrinogen 5. 1 hour after firm clotting, stand at room temperature
▪ Conditions associated with abnormal clot retraction: 6. After retraction, remove cork
o Thrombocytopenia o adhere to applicator stick
o Glanzmann thrombasthenia 7. Compute for % retraction
❖ Deficiency of GP IIb/IIIa = no
fibrinogen binding
o Hypofibrinogenemia and Abnormal
fibrinogens
❖ Difficulty of platelet to aggregate
o Multiple myeloma, Macroglobulinemia
❖ Presence of abnormal proteins that
will inhibit production of blood clot CAPILLARY FRAGILITY TEST
A. Hirschboeck (Castor Oil Method) ▪ To evaluate fragility of capillary walls
▪ Qualitative Test o if capillaries are weak, the increase in
o observe for formation of dimpling/droplet venous pressure will cause capillaries to
like serum on the surface of blood drop rupture = Petechiae
▪ Screening test for platelet function ▪ To identify the deficiency of platelets
▪ Observe for formation of dimpling/droplet like o CFT correlates with the degree of
▪ serum on the surface of blood drop Thrombocytopenia
▪ Normal Value: 15-45 minutes A. Rumple-Leede Test
Procedure: ▪ Determine the patient blood pressure
1. Place a few drops of castor oil into clean test tube ▪ Apply sphygmomanometer to forearm for 5 minutes
2. Perform skin puncture (pressure midway of Systolic and Diastolic)
3. Discard the 1st drop of blood ▪ After 15 minutes, release the pressure cuff
4. Add 1 drop of fresh blood into the test tube with ▪ Remove cuff and examine for petechiae (surface of
castor oil. Start the timer arm, dorsal of hand and finger)
5. Observe for DIMPLING ▪ Count the petechiae on an area 1 inch in diameter
o Dimpling less than 15 minutes: Thrombotic and grade
Tendency
o Dimpling greater than 45 minutes:
Hemorrhagic Tendency
B. Stefanini Method
▪ Estimate amount or degree of retraction
Procedure:
1. Extract 3 mL of blood
2. Transfer into the test tube
3. Incubate for 1 hour
4. Stand at room temperature
Result:
CR begins within 1 hour, complete within 18-24 hours
✓ Complete
✓ Partial
✓ Poor
✓ Very Poor
 TOPIC TITLE LOREM IPSUM
Mr./Ms. Professor | 2nd Semester | A.Y. 2023-2024

❖ Most Primary Hemostasis Lab Test performed:

o Platelet Count
o Est. Platelet Count
o Bleeding Time
❖ In special laboratory: Platelet Aggregometry
 TOPIC TITLE LOREM IPSUM
Ms. Aila Nell Sarmiento| 2nd Semester | A.Y. 2023-2024

OUTLINE
I. Secondary Hemostasis
II. Nomenclature of Procoagulants
III. Coagulation Groups
A. Based on Properties
i. Fibrinogen Group
ii. Prothrombin Group
iii. Contact Group
B. Based on Pathway
i. Extrinsic
ii. Intrinsic
iii. Common
IV. Coagulation Pathway
V. Thrombin Feedback

I. SECONDARY HEMOSTASIS
• Key player: COAGULATION FACTORS
o Other names: coagulation proteins,
procoagulants
o Produced mainly by the LIVER
✓ some factors are produced by
monocytes, endothelial cells,
megakaryocytes
• FORMS
o Enzymes (Zymogens -> Serine
protease/Transglutaminase)
✓ zymogen is inactivated enzyme;
when inactivated they become serine
proteases or transglutaminase
o (V and VIII) Cofactors: Enhances the
activity of enzymes
o Thrombin substrate: Factor I Fibrinogen
✓ a protein where thrombin binds to
o Plasma glycoproteins: Controls proteins that
avoid unnecessary blood clotting
✓ maintains balance in secondary
hemostasis
✓ ex of control protein: thrombomodulin
- prevents excessive production of
thrombin
II. NOMENCLATURE OF PROCOAGULANTS
• 1958, International Committee for the
Standardization of the Nomenclature of the Blood
Clotting Factors
• Named according to their order of initial
description/recovery
• Roman numerals
• “a” (activated) appears behind the numeral to denote
that the procoagulant has been activated
o Ex: Ia
o The zymogens include II, VII, IX, X, XI, XII,
and prekallikrein
o The serine proteases are IIa, VIIa, IXa, Xa,
XIa, XIIa, and kallikrein
• Factors I, II, III, IV - preferable called by their names
 TOPIC TITLE LOREM IPSUM
Ms. Aila Nell Sarmiento| 2nd Semester | A.Y. 2023-2024

• Adsorbed by BaSO4 and Al(OH)3

• VITAMIN K DEPENDENT
o Problems in Vit. K = clotting disorder
• CALCIUM DEPENDENT
• Reduced by oral anticoagulant
(Warfarin/Coumadin/Coumarin)
VITAMIN K
• A quinone found in green leafy vegetables
• Produced by intestinal bacteria:
o Bacteroides fragilis
o Escherichia coli
• Vit. K Dependent: *vit k is important in production of the ff*
o Procoagulants: II, VII, IX, X
o Regulatory Proteins: Protein C (destroys factor
V and VII), S, Z
• VIT. K IS DIFFERENT FROM POTASSIUM
• Possible causes of Vit. K deficiency:
1. Diet
2. Antibiotics
• For gamma carboxylation
o Enables the coagulation factors and
coagulation control proteins to bind ionic
calcium and cell membrane phospholipids,
especially phosphatidylserine
o Non-functional: Proteins induced by Vit. K
antagonists
• Causes of synthesis of non-functional factors:
o Vitamin K Deficiency
o Antibiotics
o Oral Anticoagulants
iii. Contact Group
• Activated via contact in foreign substances
• Members: XII, PK, HMWK, XI
• Produced in the liver
III. COAGULATION GROUPS
• Present in fresh plasma and serum
• Based on properties
• Activation: required contact with foreign surface
• Based on pathway
o in vivo
A. based on Properties
o in vitro
• Not based on roles on coagulation cascade
• VITAMIN K INDEPENDENT
• Important for LABORATORY ASSAYS
• CALCIUM DEPENDENT
1. Fibrinogen group
B. Based on Pathway
2. Vitamin K-Dependent Prothrombin group
• interdependent
3. Contact group
i. Fibrinogen Group • The pathways are not exclusive; all of them occur at
the same time
• Activated by thrombin
• They ALL work together to make fibrin clot
• Members: I,V, VIII, XIII
1. Intrinsic
• Completely consumed in clotting
2. Extrinsic
• Not present in serum – mixing studies (to identify 3. Common
missing coagulation factors i. Extrinsic Pathway
• Cleaved by thrombin • Tissue Factor Pathway
o fibrinogen converted by thrombin to fibrin clot
• Extrinsic “originating outside”
• Destroyed by plasmin
• initiated (started) by tissue factor, by which is
o plasmin: protein involved in fibrinolysis
normally outside the bloodstream or vascular system
• Found in platelets: I, V, VIII o tissue factor comes from the damaged blood
• VITAMIN K INDEPENDENT vessel lining that is found underneath the
• CALCIUM DEPENDENT vascular intima
• With highest molecular weight • Laboratory assay: Prothrombin Time
ii. Prothrombin Group ii. Intrinsic Pathway
• Members: II, VII, IX,X • Contact-Activation Pathway
• Produced in the liver • Intrinsic “originating within”
• Present in FRESH plasma and serum •
o Not present in adsorbed plasma •
 TOPIC TITLE LOREM IPSUM
Ms. Aila Nell Sarmiento| 2nd Semester | A.Y. 2023-2024

• involves coagulation factors circulating within the

bloodstream that are activated when they contact the
surface of certain cells
• Laboratory assay: Activated Partial Thromboplastin
Time (APTT)
iii. Common Pathway
● Links extrinsic and intrinsic pathways
● Final product: formation of fibrin clot

B. Intrinsic Pathway
• Factor XII activated by exposure to collagen
• Factor XIIa HMWK, & PK activate Factor IX
• IXa:VIIIa activates Factor X
• Factors involved: VIII, IX, XI, XII
• Complex formed: IXa:VIIIa (Intrinsic tenase)
o PK is activated by Factor XII to form Kall. Kall
will further activate Factor XII into Factor
XIIa. Factor XIIa will activate Factor XI to
Factor XIa. Factor XIa will activate Factor IX,
then Factor IXa will form a complex with
Factor VIII (IXa:VIIIa) which will also activate
Factor X.

IV. COAGULATION PATHWAY

A. Extrinsic Pathway
• Tissue Factor from injured blood vessel wall
activates factor VII
• TF: VIIa activates factor X
• Factors involved: TF (III), VII
• complex formed: TF:VIIa (Extrinsic tenase)
o TF activates Factor VII, they will form a
complex (TF:VIIa) that will activate Factor X.
After activation, Factor X will form a complex
with Factor V converting thrombin into
prothrombin– which is needed to convert
fibrinogen into fibrin polymer and eventually
the fibrin clot. Fibrin stabilizing factor is
Factor XIII.
 TOPIC TITLE LOREM IPSUM
Ms. Aila Nell Sarmiento| 2nd Semester | A.Y. 2023-2024

C. Common Pathway
• Both extrinsic and intrinsic pathway lead to the
activation of factor X
• Factors involved: X, V, II, I
• complex formed: Xa:Va (Prothrombinase)
• Xa:Va converts prothrombin (II) to thrombin (IIa)
• Thrombin cleaves fibrinogen (I) into fibrin & activates
factor XIII (fibrin stabilizing factor) to stabilize clot
Notes:
Thrombin is primary enzyme of secondary degree
hemodialysis
Plasmin is primary enzyme of fibrinolysis

V.THROMBIN FEEDBACK
• Mechanism or physiologic process that helps control
the degree of coagulation
• Low thrombin levels activate factors V, VIII (positive
feedback on the cascade), and XIII and induce
platelet aggregation
• When thrombin levels are high, thrombin binds to
thrombomodulin on the endothelial surface and
activates the protein C pathway
• Activated protein C and its cofactor, protein S,
inhibits factors V and VIII (negative feedback on the
cascade)

Effects of Bound Thrombomodulin to Thrombin

1. Loss of ability to activate factors V and VIII
2. Activates Protein C, leading to destruction of factors V
and VIII
3. Activates TAFI - fibrinolysis inhibitor
 MECHANISM OF SECONDARY HEMOSTASIS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

OUTLINE ❖ During coagulation, thrombin cleaves fibrinogen

generating a fibrin clot and activates factors V, VIII, XI,
I Coagulation Regulatory Mechanisms
and XIII propagating more thrombin generation.
A Tissue Factor Pathway Inhibitor (TFPI)
i 2 Steps of Coagulation Inhibition
• EC Protein C Receptor (ECPR)
B Protein C Regulatory System
o A transmembrane protein that binds protein C
i Steps of Protein C Regulatory System adjacent to the thrombomodulin-thrombin complex,
ii Protein C & S to activate PC (PC to APC).
iii Did You Know? Augments the action of thrombin-thrombomodulin at
C Antithrombin least fivefold in activating protein C to the serine
i Heparin protease APC.
ii Steps of Antithrombin • Protein S
D Other Serine Protease Inhibitors o Cofactor that binds and stabilizes Activated Protein C
(APC), is synthesized in the liver, and circulates in
plasma.
COAGULATION REGULATORY MECHANISMS
• Provide feedback loops to maintain a complex and delicate 2 Forms of Protein S
balance between abnormal thrombosis and bleeding.
Free Protein S
• Ensure localized coagulation (not systemic) and prevent Can serve as the APC cofactor
excessive clotting or thrombosis. (40%)
• Inhibitor deficiency leads to thrombosis or Bound Protein S -
-
thromboembolism that can block the blood vessels to C4bBP (60%)
cause death.
• Inhibitors (Natural Anticoagulant) Steps of Protein C Regulatory System
• Function to slow the activation of 1. Thrombin binds to thrombomodulin, activating the protein
Key Player C system.
procoagulants and suppress thrombin
production. 2. EPCR binds protein C adjacent to the thrombomodulin-
thrombin complex for activation.
• Tissue Factor Pathway Inhibitor (TFPI) 3. APC dissociates from EPCR and binds to Free Protein S.
Principal
• Antithrombin (AT) 4. APC-Protein S complex hydrolyzes and inactivates
Regulators factors Va and VIIIa, slowing or blocking thrombin
• Activated Protein C (APC)
generation and coagulation.
TISSUE FACTOR PATHWAY INHIBITOR (TFPI)
• Principal regulator of the Tissue Factor pathway or Extrinsic
pathway.
• It is synthesized primarily by endothelial cells (ECs) and is
also expressed on platelets.
• It is a Kunitz-type serine protease inhibitor and is the
principal regulator of the Tissue Factor or Extrinsic
pathway.
o Kunitz - 2: Binds to and inhibits Factor Xa.
o Kunitz - 1: Binds to and inhibits VIIa:TF complex
(Extrinsic Tenase)
• Enhanced by Protein S.
o Protein S: the cofactor of APC, is also a cofactor of
TFPI, and enhances factor Xa inhibition by TFPI
tenfold.

Steps of Coagulation Inhibition Protein C & S

1. TFPI first binds to Factor Xa and inactivates it. • Vitamin K - dependent regulator proteins.
a. Factor Xa is in the common pathway which is the • Protein C is activated when thrombin binds to
product of activation of the TF:VIIa complex. thrombomodulin on the endothelial cell surface.
2. TFPI:Xa complex binds and inactivates the cell bound • Both Inhibits Factor V and VIII to provide negative feedback
TF:VIIa complex, preventing more activation of Factor Xa. on the cascade.
Did You Know?
• Protein S-C4bBP binding is of particular interest in
inflammatory conditions because C4bBP is an acute
phase reactant.
• When the plasma C4bBP level increases, additional
protein S is bound and free protein S levels become
proportionally decreased, which may increase the risk of
PROTEIN C REGULATORY SYSTEM thrombosis.
• Activated by thrombin-thrombomodulin complex.
• Revises thrombin’s function from pro- to anticoagulant. Inflammation → ↑plasma C4bBP level → Binding of
o Thrombin: a primary enzyme of secondary protein S to C4bBP → ↓cofactor available & Protein C →
hemostasis, wherein in this method, it is used as a Thrombosis
facilitator to control its further production.
 MECHANISM OF SECONDARY HEMOSTASIS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

ANTITHROMBIN • Potent inhibitor of Factor Xa,

• Produced in the liver. requires Protein Z, calcium,
• First identified coagulation regulatory protein, and first to ZPI (Z - dependent and phospholipid.
be assayed routinely. Protease Inhibitor) • Inhibits Factor XIa,
• It is the principal inhibitor of coagulation. enhanced by heparin.
• Inhibits the serine proteases (intrinsic pathway), including • Vitamin K dependent.
thrombin (factor IIa) and factors IXa, Xa, XIa, XIIa,
prekallikrein, and plasmin. • Found in plasma and other
• Enhanced by Heparin. body organs.
• Inhibits variety of proteases.
Heparin Protein C Inhibitor • Non-specific heparin-binding
• It enhances the action of antithrombin and is a member of serpin
glycosaminoglycan family of carbohydrates. • Protects Factor Va & VIIIa.
• Available from endothelium-associated mast cell granules
or as EC Heparan Sulfate. A1-protease
Inhibits Factor XIa, and it
❖ A cofactor or known as Heparin Cofactor II (HC II). inhibitor (Alpha 1
inactivates plasmin.
Antitrypsin)
Steps of Antithrombin
1. The heparin binds to antithrombin and changes its Alpha 2- Inhibits Thrombin, Factor Xa,
conformation or binding site. Macroglobulin Kallikrein, and Plasmin
2. AT binds to thrombin, forming Thrombin-Antithrombin- • Principal inhibitor of
Heparin complex (T-AT-Heparin). Alpha 2- Fibrinolysis.
3. T-AT detaches from heparin, is known as thrombin- Antiplasmin
• Inhibits free plasmin.
antithrombin (TAT) complexes which is the one measured
in the laboratory. • Important inhibitor of
Fibrinolysis.
Plasminogen • Secreted by endothelium.
Activator Inhibitor- • Inhibits plasminogen
1 (PAI-1) activator.
• Released from EC upon
damage.

OTHER SERINE PROTEASE INHIBITORS

 VASCULAR PLATELET DISORDER
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

OUTLINE HEMANGIOMA – THROMBOCYTOPENIA SYNDROME (HTS)

I Vascular Disorders • Also known as ‘Kasabach-Merritt Syndrome.’
II Hereditary Vascular Bleeding Disorders • Hemangioma: it refers to tumor built of blood vessels, which
A Hereditary Hemorrhagic Telangiectasia may be visceral (organs), or subcutaneous (skin).
B Hemangioma - Thrombocytopenia Syndrome
C Ehlers - Danlos Syndrome • Vascular tumor where platelet is secreted, found on
III Acquired Vascular Disorders children, and newborns.
A Allergic Purpura (Henoch - Schonlein Purpura)
B Senile Purpura Giant cavernous hemangioma,
C Drug - Induced Vascular Purpura Characteristics Thrombocytopenia, and Bleeding
IV Miscellaneous Vascular Bleeding Disorders diathesis
A Paraproteinemia & Amyloidosis
B Scurvy DIC, sequestration of platelets
(through hemangioma),
Complications Microangiopathic Hemolytic
VASCULAR DISORDERS
Anemia (due to mechanical
• Problems in the integrity of the blood vessels can also lead to
damage)
bleeding or hemorrhage.
Treatment Corticosteroid, surgery
Hereditary Vascular Disorders
Mortality 30%
• Hereditary Hemorrhagic Telangiectasia
• Hemangioma Thrombocytopenia
• Ehlers - Danlos Syndrome

Acquired Vascular Disorders (Due to Allergy & Old

Age)
• Henoch - Schonlein Purpura
• Paraproteinemia & Amyloidosis
EHLERS – DANLOS SYNDROME (EDS)
• Senile Purpura • Characterized by hyperextensible skin, hypermobile joint,
• Drug - Induced Vascular Purpura fragile tissues, and bleeding tendency.
• Platelet abnormalities may also be present.
HEREDITARY VASCULAR BLEEDING DISORDERS • Due to defects in collagen production, structure, cross-
linking, leading to inadequacy. Hence, a major problem in
HEREDITARY HEMORRHAGIC TELANGIECTASIA (HHT) elasticity.
• It is also known as ‘Rendu - Osler - Weber Syndrome’, and
‘Spider Veins.’
• Telangiectasias
o Dilated superficial blood vessels that create small,
focal red lesions.
o Most obvious on the face, lips, tongue, conjunctiva,
nasal mucosa, fingers, toes, and trunk & under the
tongue.
Thin - walled blood vessels
with discontinuous endothelium,
Characteristics inadequate smooth muscle,
and/or missing elastin around
stroma ACQUIRED VASCULAR DISORDERS
• Due to drug exposure or allergy and focuses on
Manifestations Telangiectasia Immunoglobulin IgA.
Universal
Findings
Epistaxis (nosebleed) ALLERGIC PURPURA (HENOCH – SCHÖNLEIN PURPURA)
• Also known as ‘Anaphylactoid Purpura.’
• Not caused by thrombocytopenia.
• Characterized by skin rash (palpable purpura) and edema.
• Henoch - Schönlein Purpura: an IgA - mediated vasculitis,
hence, becomes deposited in the blood vessels.
• Most common in children, 3 - 7 yrs. old.
 VASCULAR PLATELET DISORDER
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

o It causes hyperviscosity, affecting blood flow.

• Amyloid
o Fibrous protein consists of rigid, linear, non - branching,
aggregated fibrils.
o It can be deposited in vascular walls, causing
thrombosis, and hemorrhage.
• Primary affect platelet function.
SCURVY
• Appearance of petechiae due to decreased synthesis of
collagen.
• Caused by deficiency of Vitamin C (promotes collagen
production).

SENILE PURPURA
• Found in elderly, particularly in the areas exposed to sunlight.
• Caused by lack of collagen support for small blood vessels
and loss of subcutaneous fat and elastic fibers.
• No other bleeding manifestations.
Lesions
• Extensor surfaces of the forearms
• Backs of the hands
• Face
• Neck

DRUG – INDUCED VASCULAR PURPURA

Vascular Purpura Caused By
• Aspirin: it inhibits Thromboxane A2 (Eicosanoid
Pathway)
• Warfarin: Vitamin Depletion
• Barbiturates
• Diuretics
• Digoxin
• Methyldopa
• Sulfonamides
• Iodides
Formation of antibodies immune
Mechanism complexes and damages in vessel wall
permeability

MISCELLANEOUS VASCULAR BLEEDING DISORDERS

PARAPROTEINEMIA & AMYLOIDOSIS

• Paraproteins
o Wherein immunoglobulin fragments produced, that
increase viscosity in multiple myeloma.
 LEC #13 HEMORRHAGIC DISORDERS
Ms. Aila Nell Sarmiento | 2nd Semester | A.Y. 2023-2024

OUTLINE C. Acquired Bleeding Disorders

• Acquired: Due to exposure or something
patient have developed later on through life
• Clinical Manifestations first occur in:
1. Adulthood
2. Associated with another disease
(secondary)
3. Not found in relatives
4. due to drug exposures
• Associated Diseases:
✓ Liver Disease
✓ Vitamin K deficiency
✓ Renal failure
• LABORATORY TEST:
✓ CBC (Complete Blood Count)
I. BLEEDING SYMPTOMS ✓ PT (Prothrombin time)
• Hemorrhage - excessive bleeding that requires ✓ PTT (Partial Thromboplastin Time)
medical or physical intervention ✓ Fibrinogen and Thrombin time
• Forms of bleeding
✓ Local (single location) D. Congenital/Hereditary Bleeding Disorders
▪ May be due to: injury, infection or • Hereditary/Congenital: Due to mutation,
isolated blood vessel defect inherited a gene mutation from either parent
▪ Example: Patient has physical • Uncommon, occurs in 1 in 100 individuals
trauma that resulted to bleeding • Diagnosed in
✓ General (multiple sites) ✓ Infants, young children
▪ spontaneous and recurring ✓ With relatives who has similar symptoms
▪ The effects are systemic and • Leads to repeated hemorrhages
widespread due to manifestations ✓ Spontaneous
found throughout the body ✓ After minor injury
➢ Requires intervention and transfusion • Mild disorders: Symptoms are experienced in
➢ May be due to: adulthood
1. Disorder of blood vessel • Most common congenital disorders:
2. Disorder of platelets and 1. Von Willebrand Factor
thrombocytopenia 2. Factor VIII and IX Deficiency
3. Coagulation Factor deficiency and 3. Platelet function disorders
uncontrolled fibrinolysis II. ACQUIRED COAGULOPATHIES
1. Trauma-induced Coagulopathy
A. Mucocutaneous Hemorrhage 2. Liver Disease
• Skin and body orifice (Superficial) 3. Chronic Renal Failure and Hemorrhage
• Symptoms include: (skin manifestations) 4. Vitamin K Deficiency and Hemorrhage
✓ Purpura 5. Auto anti-Factor VIII Inhibitor and Acquired
✓ Petechiae Hemophilia
✓ Ecchymoses/bruises 6. Acquired Von Willebrand Disease
✓ Menorrhagia 7. Disseminated Intravascular Coagulation
✓ Hematemesis A. Trauma-induced Coagulopathy
• Mostly associated with platelet and vascular • Accounts for most instances of fatal
disorders hemorrhage
B. Anatomic Hemorrhage • Trauma-induced: may be self-inflicted,
• Soft tissue, muscles, joints, deep tissue felonious, or combat-related
• Excessive bleeding after: • Coagulopathy: any single or multiple coagulation
✓ Minor trauma factor or platelet deficiency
✓ Dental extraction • Triggered by injury-related acute inflammation and
✓ Surgical Procedure other elements of systemic shock. This leads to
• Mostly associated with coagulation factor the acute reduction of ADAMTS13.
deficiencies
 LEC #13 HEMORRHAGIC DISORDERS
Ms. Aila Nell Sarmiento | 2nd Semester | A.Y. 2023-2024

• Other Effects: Tissues Factor release,

coagulation factor activation, loss of coagulation ✓ Platelet aggregation and secretion are also
control proteins, and hyperfibrinolysis. suppressed
• Treatment: Massive transfusion iii. DIC/ Disseminated Intravascular Coagulation
✓ RBCs, plasma, platelet concentrate • Complication of liver disease
• May be acute/uncompensated or
A. Liver Disease Chronic/compensated
• Most common acquired clotting factor deficiency • Cause by:
• Liver: Produces nearly all of the plasma ✓ decreased production of regulatory
coagulation factors and regulatory proteins antithrombin, protein C and protein S
▪ Without a healthy liver there is a lack of ▪ They prevent excessive
coagulation factors and regulatory coagulation in the body. If there
proteins. are no regulatory proteins it will
• Liver Disease: Alters production Vitamin K- induce DIC
dependent factors ✓ Release of activated procoagulants from
▪ without vitamin k the carboxylation can’t degenerating liver cells
be performed ✓ Failure of liver to clear activated
✓ Produces des-y-carboxyl coagulation factors coagulation factors
▪ Patient has impaired vitamin k-dependent • LABORATORY TEST (Acute DIC)
factors due to the facilitating organ, which ✓ PT, PTT, Thrombin time
is the liver, is not functioning properly ✓ Fibrinogen: less than 100 mg/dL
• Markers of Liver Disease ✓ FDP, D-dimer
1. Factor V - most specific marker of Liver Function • LABORATORY TEST (Chronic DIC)
Test ✓ D-dimer is the ONLY ABNORMAL TEST
➢ Not Vitamin K dependent and dietary B. Chronic Renal Failure and Hemorrhage
Vitamin K deficiency • Associated with platelet dysfunction and mild
2. Factor VII - sensitive early marker to moderate mucocutaneous bleeding
3. fibrinogen (acute phase reactant) • Platelet adhesion and aggregation are
➢ Early/mild liver disease: elevated suppressed because of platelet counting by
➢ Moderate/severe liver disease: guanidinosuccinic acid or dialyzable phenolic
dysfibrinogenemia compounds.
➢ End-stage/Liver failure: Decreased; less • Bleeding: due to anemia and thrombocytopenia
than 100 mg/dL i. Hemostasis activation syndromes (DIC, HUS, TTP)
4. Von Willebrand Factor, Factor VIII and Factor XIII • reduces glomerular function through fibrin
(other acute phase reactants) deposition
➢ Unaffected or elevated in mild to • initially thrombotic disorders but also causes
moderate liver disease thrombocytopenia and mucocutaneous bleeding
• TREATMENT: Fresh Frozen Plasma • TREATMENT:
i. Dysfibrinogenemia 1. Dialysis
• presence of fibrinogen with excessive sialic 2. RBC Transfusion
acid residues caused by liver disease 3. Erythropoietin therapy
• Symptoms: Mild tissue bleeding 4. Desmopressin acetate
• Examination: Thrombin time, Reptilase time ii. Nephrotic Syndrome and Hemorrhage
➢ Difference between Thrombin time and • low molecular weight proteins are lost
Reptilase time is that Reptilase time is not because of increase glomerular permeability
affected by heparin ✓ includes factors II, VII, IX, X, XII
ii. Moderate Thrombocytopenia (2,7,9,10,12), protein C, antithrombin
• Due to • Increased glomerular permeability associated
✓ Shortened platelet survival with:
✓ Sequestration associated with portal hypertension 1. Chronic glomerulonephritis
and hepatosplenomegaly 2. Diabetic glomerulosclerosis
▪ Spleen is near the Liver 3. SLE
▪ 30% is the normal platelet count in spleen 4. Amyloidosis
but in hepatosplenomegaly more than 5. Renal vein thrombosis
30% is there.
✓ Alcoholic toxicity suppresses platelet production
 LEC #13 HEMORRHAGIC DISORDERS
Ms. Aila Nell Sarmiento | 2nd Semester | A.Y. 2023-2024

C. Vitamin K Deficiency and Hemorrhage • Protein structure: 4 domains (A to D)

• Sources of Vitamin K: Green leafy vegetables ✓ Domain A: supports a receptor site for
• Fat soluble, requires bile salts for absorption collagen and a binding site (ligand) for
• Causes platelet receptor GP Ib/IX/V and heparin
1. Biliary duct obstruction ✓ Domain C: provides a site that binds to
2. Fat malabsorption platelet receptor GP IIb/IIIa BARIUAN,V.R
3. Chronic diarrhea & BADIDLES, L.C. 3
4. Use of broad spectrum ✓ Domain D: provides the carrier site of
➢ broad spectrum could kill the normal flora factor VIII
in the gut leading to no vitamin k ▪ von willebrand factor protects
5. Breastfeeding in Newborns factor VIII from proteolysis,
➢ Passively acquired maternal antibodies prolonging their half life
delay the establishment of gut flora • Function is to mediate platelet adhesion and
6. Coumadin overdose aggregation
➢ includes Brodifacoum “super warfarin” -
rodenticide (lead to Vitamin K deficiency).
• Additional Causes of Vitamin K Deficiency
1. Sterile intestine of newborns
2. Minimal concentration of Vitamin K in human milk
3. Breastfeeding: maternal antibodies delay
establishment of normal flora

D. Acquired Von Willebrand Disease

Von Willebrand Disease
• Named after Dr. Erik Adolf von Willebrand, a
Finnish Pediatrician who first described the
condition.
• Was first termed as ‘Pseudohemophilia’
• most prevalent congenital bleeding disorder
• Resulted from quantitative and qualitative
abnormalities of Von Willebrand Factor
• Mucocutaneous disorder
• Soft tissue bleeding
• Reduced platelet adhesion
• Blood product for transfusion: Cryoprecipitated
AHF
• encoded by an autosomal gene on
chromosome 12
• Has 3 subtypes:
✓ subtype 1 and 2 DOMINANT pattern of
inheritance EXCEPT “2N” it is autosomal i. Type 1 shortage of von willebrand factor (MILD)
recessive • Partial quantitative decrease of normal von
✓ subtype 3 RECESSIVE pattern of willebrand factor and Factor VIII
inheritance • Most common and mildest form of von
Basic Biology and Function of von willebrand factor willebrand disease
• Multimeric glycoprotein • SDS polyacrylamide gel: small, intermediate and
• von willebrand factor gene: Chromosome 12 large multimers present
• Synthesize in the ii. Type 2 (Flawed von willebrand factor)
✓ endoplasmic reticulum if endothelial cells • Primary qualitative defects of von Willebrand
✓ Megakaryocytes factor
• Stored in the • Type 2: 15-20% of patients
✓ Weibel-Palade bodies • can be either autosomal dominant or recessive
✓ Alpha granules of platelets iii. Type 2A
• Inherited as autosomal dominant
• Autosomal mutations in A2 structural domain
of von willebrand factor
 LEC #13 HEMORRHAGIC DISORDERS
Ms. Aila Nell Sarmiento | 2nd Semester | A.Y. 2023-2024

✓ more susceptible to ADAMTS13 • LABORATORY RESULTS

✓ Increases small molecular-weight ✓ von willebrand factor level: <1%
multimers ✓ Factor VIII clotting activity: 2%-8%
✓ less platelet adhesion ✓ Bleeding time >15 minutes
• LABORATORY RESULTS:
✓ von willebrand factor:Ag = Normal/Slightly
reduced
✓ von willebrand factor activity: Reduced
iv. Type 2B
• Inherited as autosomal dominant trait
• <5% of von willebrand disease patients
• mutations in the A1 domain which increases the
affinity of von willebrand factor to Glycoprotein
Ib/IX/V = “gain-of-function” mutation
• leads to spontaneous binding to resting platelets
• decrease of high molecular-weight von
willebrand factors and platelets
• SDS polyacrylamide gel: small and intermediate
multimers are present
• platelet-type von willebrand disease/pseudo-
von willebrand disease: same mechanism, but
the mutation is with Glycoprotein Ib of platelets
v. Type 2M
• Characterized by possesses poor platelet
receptor binding
• SDS polyacrylamide gel electrophoresis: small
intermediate and large multimers are present
• Often incorrectly identified asa Type 1 or
subtype 2A
✓ differentiating test: von willebrand factor
ristocetin cofactor assay
vi. Type 2N
(Normandy variant or autosomal hemophilia)
• mode of inheritance: autosomal recessive E. Hemophilia
• markedly decreased affinity of von Willebrand • Congenital single-factor deficiencies
factor for factor VIII, resulting in Factor VIII levels • Characterized by anatomic soft tissue
reduced to usually around 5% of reference range bleeding
• von willebrand factor antigen concentration • Second to von willebrand disease in
(von willebrand factor:Ag) and ristocetin prevalence
cofactor activity are usually normal • Affects mostly males
vii. Type 3 1. Hemophilia A: Factor VIII (8) deficiency
• Complete lack of von willebrand factor (more 2. Hemophilia B: Factor IX (9) deficiency
severe) 3. Hemophilia C: Factor XI (11) deficiency
• “Null allele” von willebrand factor gene ✓ aka Rosenthal syndrome
translation or deletion mutation Genetics
• Absence of von willebrand factor from both • Most people with hemophilia A or B are males
platelets and endothelial cells, and a lack of • Women are carries
response to desmopressin acetate ✓ Defective gene can be passed
• Factor VIII is also absent/diminished down to their MALE offsprings
• most rare form of von willebrand disease • Females: XX chromosome
• Produces severe mucocutaneous and anatomic • Males: XY Chromosome
hemorrhage
 LEC #13 HEMORRHAGIC DISORDERS
Ms. Aila Nell Sarmiento | 2nd Semester | A.Y. 2023-2024

Father with hemophilia + Mother without hemophilia

➢ 50% chance of affected/carrier female
offsprings
➢ 50% chance of non-affected male
offsprings

Father without hemophilia + Mother with hemophilia

➢ 50% chance of affected/carrier female
➢ 100% chance of affected male offspring
Father with hemophilia + Mother with hemophilia
➢ 50% chance of affected female offsprings
➢ 50% chance of affected male offsprings
➢ Rare occurrence
 LEC #13 HEMORRHAGIC DISORDERS
Ms. Aila Nell Sarmiento | 2nd Semester | A.Y. 2023-2024

Signs and Symptoms iv. Factor VII deficiency

• Many to large or deep bruises • Rare (1:500,000) autosomal recessive deficiency
• Joint pain and swelling caused by internal • Exhibit little correlation between the bleeding
bleeding risk and the factor activity
• Bleeding within muscles • <1% activity: severe bleeding similar to
• Blood in urine or stool hemophilias
• Prolonged bleeding from cuts or injuries, or after • >5% activity: mild bleeding that is often localized
surgery or tooth extraction to mucous membranes
v. Factor X deficiency
Treatment • Inherited as an autosomal recessive trait, may
• factor VIII/IX concentrates (may be human plasma also be acquired
derived or recombinant ) • Acquired: amyloidosis, paraproteinemia,
• Cryoprecipitate antifungal drug therapy
• Mild hemophilia: Desmopressin acetate (DDAVP, vi. Factor XIII deficiency
1-deamino-8-D-arginine vasopressin) • Rare (1 per 1 million) autosomal recessive
✓ helps the body release factor VIII disorder.
that is stored within the lining of • Acquired factor XIII deficiency: Henoch-
blood vessels. Schonlein purpura, erosive gastritis, and
leukemia
i. Hemophilia A • LABORATORY RESULTS:
• Classic Hemophilia: lack of blood clotting factor ✓ PT, APTT, Thrombin time: NORMAL
VIII (8) ✓ Poor wound healing and anatomic
ii. Hemophilia B bleeding
• “Christmas disease” (Stephen Christmas – • Screening Test: 5M urea assay
first patient) vii. Acquired Factor XI deficiency
• Hereditary Deficiency of Factor IX (9) • Occurs in patients who develop inhibitors factor XI
iii. Hemophilia C (Rosenthal Syndrome) • Observed in patients with SLE and other
• Factor XI (11) deficiency immunologic disease
• Autosomal dominant trait • Common finding in patients with Noonan
• Mild to moderate bleeding symptoms syndrome, which is characterized by congenital
• Predominantly occurs in Jews of Ashkenazi cardiac abnormalities, short stature, and mental
descent retardation
• To differentiate Hemophilia C from Hemophilia A:
✓ Absence of bleeding into joints
and muscle
✓ Occurrence in individuals of
either sex

F. Other Congenital Single-factor deficiencies

i. Factor I deficiency
• Deficiency of Factor I (fibrinogen) may be
inherited (afibrinogenemia) or acquired
ii. Factor II deficiency
• Deficiency of Factor II (prothrombin) leads to
hypoprothrombinemia.
• The deficiency may be acquired due to
prothrombin antibodies
iii. Factor V deficiency
• Also known as proaccelerin or accelerator
globulin or labile factor
• Rare hemorrhagic tendency known as Owren’s
disease or parahemophilia
LEC #13 HEMORRHAGIC DISORDERS
Ms. Aila Nell Sarmiento | 2nd Semester | A.Y. 2023-2024
 MONITORING ANTICOAGULANT THERAPY
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

OUTLINE Antithrombotic Drugs

I Thrombosis
II Coumadin (Warfarin) Therapy Suppress coagulation and
A Coumadin Prophylaxis and Therapy Anticoagulant
reduce thrombin formation
B Warfarin Therapy Monitored Using the PT Assay
C Warfarin INR Therapeutic Range Antiplatelet Drugs suppress platelet activation
D Reversing Coumadin
III Heparin Therapy disperse or reduce existing clots
A Unfractionated Heparin (UH) Fibrinolytics clogging veins and arteries;
i Monitoring UH Therapy enhances fibrinolysis
ii Limitations of PTT Monitoring of UH
iii Monitoring UH Using ACT
iv Reversal of UFH Overdose COUMADIN / WARFARIN THERAPY
B Low Molecular Weight Heparin (LMWH) • Oral anticoagulant therapy (OAT)
i Advantages of LMWH Over UH o New Direct - Acting Oral Anticoagulants: rivaroxaban,
ii Monitoring of LMWH apixaban, edoxaban, and dabigatran (brands).
IV Direct Thrombin Inhibitors • Vitamin K Antagonist: inhibits the vitamin K dependent
V Antiplatelet Drugs factors.
• Prevents gamma carboxylation of coagulation factors by
THROMBOSIS Vitamin K.
• Pathological formation of blood clots in veins or arteries that o Gamma Carboxylation: Enables these coagulation
factors and coagulation control proteins to bind ionic
obstruct flow and cause tissue ischemia and necrosis. calcium and cell membrane phospholipids, especially
Table14.1 Anticoagulants Included phosphatidylserine.
o Proteins Induced by Vitamin K Antagonists (PIVKAs):
Low they are known as non - functional or non - carboxylated
Unfractionat
Molecular proteins.
Coumadin ed
Weight
(Warfarin) Heparin
Heparin Vitamin K
(UFH) II, VII, IX, X, Protein C, S and Z
(LMWH) dependent
Admini Subcutaneo Vitamin K is
Oral IV green tea, avocados, and green leafy
stration us Concentrated
vegetables
Catalyzes in
inhibition of Catalyzes Produced in Gut Flora
Vitamin K
Action thrombin, Xa inhibition of
antagonist
& Xa by AT
IXa by AT
Effect Slow acting Immediate Immediate
Longer than
Duratio UFH,
Long Short
n shorter than
warfarin
• Monitorin
g usually
Test(s) APTT, ACT not
for (point of required.
PT
monitor care), anti - • If needed,
ing factor Xa anti-factor
Xa should
be used.
Decreases
Requires AT APTT is
production
Other to be insensitive COUMADIN PROPHYLAXIS & THERAPY
of II, VII, IX,
effective to LMWH
X • Administered prophylactically to prevent transient ischemic
Acetaminophen, aspirin (acetyl salicylic acid) and ibuprofen attack and strokes in patients with nonvalvular atrial
may inhibit platelet activation by blocking cyclooxygenase fibrillation.
o Ischemia: absence or failure of oxygen delivery to the
tissue.
• Prevent venous thromboembolism after trauma, orthopedic
surgery, and general surgery, and in a number of chronic
medical conditions.
• Administered therapeutically after acute myocardial
infarction (AMI) if the event is complicated by congestive
heart failure or coronary insufficiency and to control clotting in
patients with mechanical heart valves.
 MONITORING ANTICOAGULANT THERAPY
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

Table 14.3 Recommendation for Reversal of Coumadin

Begins with 5mg daily oral Overdose Based on INR and Bleeding
Coumadin Regimen
dose
Bleeding INR Intervention
Decreased In Reduce dosage or omit one
Coumadin (Vitamin VII → IX → X → II 3-5
dose, monitor INR frequently
K) Therapy
Omit Coumadin, monitor INR
Approximately 5 days after frequently, consider oral
Full Anticoagulant
the start of Coumadin No Significant 5-9
vitamin K (≤5mg) if high risk
Effect Bleeding
Therapy for bleeding (surgery)
Stop Coumadin, give 5 -
WARFARIN THERAPY (PROTHROMBIN TIME ASSAY) >9 10mg oral vitamin K, monitor
• Anticoagulant becomes therapeutic only when the activity of INR frequently
Factor II and X decreases less than 50% in 5 days. • Stop Coumadin, give 10
Tissue Factor, Phospholipids, Ionic mg vitamin K by
PT Reagents intravenous push (may
Calcium
Serious Any repeat every 12hr)
PT Prolong within 6 - 8 hours Bleeding INR • Give thawed fresh - frozen
1st PT After 24hrs, daily, until the target plasma, prothrombin
Performed range is achieved complex concentrate, or
recombinant factor VIIa
Monitoring Every 4 - 12 weeks until completion of
Same as for serious
Continues therapy Life -
Any bleeding, except stronger
Threatening
Warfarin INR therapeutic Range INR indication for recombinant
Bleeding
• Its value will standardize the different hospitals that utilize factor VIIa
different reagents and machines.
HEPARIN THERAPY
2 - 3 or 2.5 - 3.5 • Uses Antithrombin (AT).
Adjust Dosage To • Coagulation Affected: Thrombin, IX, X, XI, XII
(common)
INR’s >4 Hemorrhage Procedure for Antithrombin (AT)
5
Immediate 1. The heparin binds to antithrombin and changes its
Communication conformation or binding site.
Dosage adjustments are made conservatively because the INR 2. AT binds to thrombin, forming Thrombin-
requires 4 - 7 days to stabilize, but an elevated INR Antithrombin-Heparin complex (T-AT-Heparin).
accompanied by the symptoms of anatomic is a medical 3. T-AT detaches from heparin, is known as thrombin-
emergency. antithrombin (TAT) complexes which is the one
measured in the laboratory.
Table 14.2 International Normalized Ratio (INR) Range
INR Conditions Associated
2.0 - Deep Vein Thrombosis (DVT), High Risk
2.5 Surgery
2.0 -
Hip Surgery, Femur Fracture
3.0
2.0 - DVT, Pulmonary Embolism, Transient Ischemic
3.0 Attack
2.5 -
Mechanical Heart Valve UNFRACTIONATED HEPARIN (UH)
3.5
Recurrent Deep Vein Thrombosis and • Sulfated glycosaminoglycans extracted from porcine
2.0 - mucosa.
Pulmonary Embolism, Myocardial Infarction,
4.5 • Binds plasma antithrombin, and inactivates the Serine
Arterial Disease
protease (IIa, IXa, Xa, XIa, XIIa).
REVERSING COUMADIN
Molecular
• Treatment for Warfarin Toxicity: Vitamin K Administration 3,000 - 30,000D
Weight
Administration Intravenous (IV)
• Venous Thromboembolism
• Acute Myocardial Infarction
• Prevent reocclusioon after Stent
Used into Treat
Placement
• Maintain Vascular Patency During
Cardiopulmonary Bypass
 MONITORING ANTICOAGULANT THERAPY
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

5000 to 10,000U and then Monitoring UH Using ACT

Initially Infused • Activated Clotting Time: is a 1966 modification of the Lee -
adjusted later on
White Whole - Blood Clotting Time.
Discontinued after surgery or is • Utilizes 2 mL tubes with 12mg of diatomaceous earth.
Therapy switched to oral AC to reduce the • Invert the tube until clot forms.
risk for thrombotic events
Median of normal
UFH THERAPY Monitoring (partial thromboplastin time) ACT
98 seconds

blood is collected before therapy is • 200 to 240 secs (DVT)

1st Sample Heparin is
begun to check baseline • 400secs in CABG
Administered to
• Point of care test during
• Indicates LAC/CF deficiency. Yield Results Of
Prolonged Cardiac Surgery
• Proceed to chromogenic anti -
Baseline
factor Xa heparin assay. Reversal of UFH Overdose
Collect after 4 - 6 hours on therapy • Use protamine sulfate.
2nd Sample initiation (this time the result should fall • Derived from salmon sperm.
within therapeutic range) • It neutralizes UFH at a ratio of 100U/mg.
Blood • Shortens APTT/ACT.
collected every 24 hrs
Collection LOW MOLECULAR WEIGHT HEPARIN (LMWH)
• Prepared From UFH Using:
o Chemical (enoxaparin)
Precaution o Enzymatic (tinzaparin)
• A 40% decrease in platelet count, suggests Heparin • Provide less thrombin - antithrombin bridging but same
Induced Thrombocytopenia (HIT) factor Xa neutralization.
• Useful among pregnant women at risk of VTE because
• Treatment must be stopped and replaced by Direct
warfarin causes birth defects.
Thrombin Inhibitor (DTI) Therapy • It replaces Coumadin in patients before surgery because
of shorter half - life.
Limitations of PTT Monitoring of UH
Molecular
Shortened Result 4500 to 5000D (⅓ of UFH)
Weight
• Inflammation Administration Subcutaneous injection
o Fibrinogen greater than 500 mg/dl, factor VIII Prophylaxis For Surgery and trauma
activity greater than 190U/dl.
o PTT shortens, less sensitive to heparin. Treatment VTE
• PF4 Advantages of LMWH Over UH
o Neutralize heparin. • Decreased “Heparin Resistance”
o PTT results are shortened in specimens after 1 o Pharmacokinetics of UH are influenced by its bindings
hour of collection. to plasma protein, endothelial cell surfaces,
o Remedy: Centrifuge the sample and remove the macrophages, and other acute phase reactants.
Platelet Poor Plasma within 1hr of collection. o LMWH has decreased binding to non - anticoagulant
related plasma proteins.
• No Need for Laboratory Monitoring
Prolonged Result o When given on a weight - adjusted basis, the LMWH
anticoagulant response is predictable and
• Hypofibrinogenemia reproducible.
• Factor Deficiencies • Higher Bioavailability
• LAC • Longer Plasma Half Life
• Less Inhibition of Platelet Function: potentially less
• Presence of FDP bleeding risk
• Paraproteins • Lower Incidence of Thrombocytopenia and Thrombosis
(HIT Syndrome)
Table 14.4 International Normalized Ratio (INR) Range o Less interaction with platelet factor 4.
o Fewer heparin - dependent IgG antibodies.
Chromogenic Anti - Xa
PTT
Assay Monitoring of LMWH
Preferred by physicians for Replacement for routine • Unnecessary in majority of patients
high-risk patients clinical use • May be Useful in Specific Instances:
Responds to all plasma - Reflects only antithrombin o Accumulation due to renal insufficiency (creatinine
>4.0 mg/dL).
based heparin activities - Xa binding o Major bleeding risk factors
Facilities: test upon • aPTT not useful due to low anti - IIa activity of LMWH.
Routine UFH monitoring
physician’s monitoring • Assay of choice: Anti - Factor Xa Assay
 MONITORING ANTICOAGULANT THERAPY
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

DIRECT THROMBIN INHIBITORS

• Argatroban
• Bivalirudin
o Recombinant Analogue of Leech Saliva - Hirudin.
o Ecarin - enzyme extracted from Echis carinatus venom
that converts prothrombin to intermediate
meizothrombin, which converts fibrinogen to fibrin.
o Potential alternative for assaying argatroban and
bivalirudin
• Dabigatran: Oral Direct Thrombin inhibitor
• These drugs does not require antithrombin, and directly
inhibit Factor II.
ANTIPLATELET DRUGS (3 MECHANISMS)
• These 3 mechanisms are related to platelet aggregation.
• Aggregometry: reference method for determining aspirin
and clopidogrel responses.
• Irreversibly Acetylates and Inactivates Cyclooxygenase:
Aspirin, which prevents the formation of Thromboxane A2.
• Bind The Adenosine Diphosphate (ADP) Receptor, P2Y12
o Clopidogrel (irreversible)
o Prasugrel (irreversible)
o Ticagrelor (reversible)
• Bind the fibrinogen binding site, Glycoprotein (GP) IIb/IIIa
o Intravenous abciximab
o Eptifibatide
o Tirofiban
 MECHANISM OF FIBRINOLYSIS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

OUTLINE 3. Plasmin slowly digests fibrin to form fibrin degradation

I Introduction products (FDPs).
II Fibrinolysis a. Degradation Products: X, Y, D, E, and D - Dimer.
A Steps of Fibrinolysis
B Regulators REGULATORS
C Fibrinolysis Pathway & Inhibitors • It helps to ensure that there is no excessive fibrinolysis or
III Action of Fibrinolytic Proteins no inadequate fibrinolysis.
IV Structure of Fibrinogen
V Formation of Fibrin Clot a2 - antiplasmin Neutralizes free plasmin
VI Fibrinolysis: Degradation of Fibrinogen & Fibrin by
Plasmin Neutralizes Tissue
VII Fibrin Degradation Product
Plasminogen Activator
Plasminogen Activator
VIII Pathologic Fibrinolysis Inhibitor - 1 (PAI-1)
(TPA)
A Primary Fibrinolysis
B Secondary Fibrinolysis Thrombin Activatable Prevents the binding of
IX Fibrinolysis Laboratory Tests Fibrinolysis Inhibitor plasminogen, plasmin, &
A Whole Blood Clot Lysis Time (WBCLT)
B Euglobulin Clot Lysis Time Test
(TAFI) TPA
C Gel Formation Tests for Fibrinolysis
i Protamine Sulfate Test FIBRINOLYSIS PATHWAY & INHIBITORS
ii Ethanol Gelation Test
D Primary Vs. Secondary
E D - Dimer
i Quantitative D - Dimer
ii Qualitative D - Dimer
F Fibrin Degradation Products Immunoassay
G Plasminogen Chromogenic Substrate Assay
H Tissue Plasminogen Activator Assay
i Tissue Plasminogen Activator Activity
I Plasminogen Activator Inhibitor - 1 Assay
i Enzyme Immunoassay

INTRODUCTION
• Primary Hemostasis: it focuses on platelet function,
adhesion, aggregation, and secretion of granules that start
the clot formation.
• Secondary Hemostasis: focus on coagulation factors, Figure 9.1 Fibrinolysis Pathway & Inhibitors
coagulation cascade (intrinsic, extrinsic, and common 1. The fibrinogen is converted into fibrin because of
pathway), laboratory test that helps identify factor prothrombin and activated antithrombin during secondary
deficiencies. hemostasis.
• Clot Dissolution (Fibrinolysis): ensures that there is no 2. Afterwards, the binding of TPA & plasminogen into the
thrombosis and abnormal clot formation that can block the fibrin clot.
blood vessels which can lead to Thromboembolism. 3. The TPA will convert or activate plasminogen into
plasmin.
FIBRINOLYSIS 4. The plasmin will degrade the fibrin into fragments X, Y, D,
• Final stage of hemostatic activation. E, and D-dimer.
• Hydrolysis of fibrin by plasmin. 5. The regulators, a2-antiplasmin that inactivates free
❖ In fibrinolysis, the key player is plasmin which plasmin, Plasminogen Activator Inhibitor - 1 which
cleaves fibrin into fibrin degradation products. neutralizes TPA, and Thrombin Activatable Fibrinolysis
❖ In Secondary Hemostasis, the last protein that cleaves Inhibitor (TAFI) which prevents the binding of
the fibrinogen into fibrin clot is the thrombin activated plasminogen, plasmin, and TPA into the fibrin clot wherein this
from prothrombin. kind of protects the fibrin clot from fibrinolysis.
• Incorporation of fibrinolytic proteins begins as early as
during clot formation. ACTION OF FIBRINOLYTIC PROTEINS
❖ Early incorporation of fibrinolytic proteins can be seen
already in the fibrinogen or fibrin to help localize
fibrinolysis and prevent excessive fibrinolysis in the
circulation.
bleeding because of fibrinogen
Excessive
consumption and/or premature clot
Fibrinolysis
lysis.
Inadequate
Clot extension and thrombosis
Fibrinolysis

STEPS OF FIBRINOLYSIS
1. Plasminogen and Tissue Plasminogen Activator (TPA)
are bound to fibrin during coagulation.
a. Plasminogen: Inactive form of plasmin.
2. TPA converts bound plasminogen to plasmin.
 MECHANISM OF FIBRINOLYSIS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

3 Domains
Figure 9.2 Schematic Diagram of the Action of 1 Central E
Fibrinolytic Proteins Domain
-
• A (Top): Tissue Plasminogen Activator (TPA) activates ❖ Made up of peptide bonds
plasminogen to the serine protease, plasmin. TPA is (Alpha, Beta, and Gamma
inhibited by plasminogen activator inhibitor-1 (PAI- 2 D Domains peptide bonds).
1). α2-Antiplasmin (AP) rapidly inactivates free ❖ Bonded by Disulfide bond.
plasmin.
FORMATION OF FIBRIN CLOT
• B (Bottom): known as competitive binding, fibrinolytic
proteins TPA, plasminogen, and plasmin bind to C-
terminal lysine residues (Lys) of fibrin during clotting.
Thrombin activatable fibrinolysis inhibitor (TAI)
inhibits fibrinolysis by removing the C-terminal Lys
from fibrin, thereby reducing binding of fibrinolytic
proteins. AP N-terminus is bound to fibrin by FXIlla
cross-linking. The AP C-terminus Lys competes with
fibrin C-terminus Lys to bind plasmin, which will
inactivate it.

• Lysine residues serve as the

binding site for TPA,
Plasminogen or Plasmin.
Lysine • Important since it is also a part Figure 9.4 Formation of a Stabilized Fibrin Clot
of the mechanism on how why • One set of domains is bound to another set of domains until it
TAFI can protect the clots forms a stabilized polymer of fibrin to serve as a fibrin clot.
from excessive fibrinolysis. • When plasmin acts on a fibrin polymer or fibrin clot, it will
now cleave fibrin into different sites, forming fibrin
TAFI removes the lysine degradation products.
Thrombin residue from the fibrin clot, in
Steps to Form Fibrin Clot
Activatable which TPA, Plasminogen, and
Fibrinolysis Plasmin cannot bind to the 1. Fibrinogen is cleaved by Thrombin and forms Fibrin
Inhibitor fibrin clot that will result to Monomer.
excessive fibrinolysis 2. Fibrin Monomers bind side by side to form Fibrin
• Acts as a competitor. Polymer.
• It consumes and neutralizes 3. Fibrin Polymer will be cross-linked with Factor XIII for
the plasmin and prevent stabilization.
excessive fibrinolysis. 4. Cross-linked Fibrin clot.
• Only has lysine residue,
α2-antiplasmin
hence will bind to plasmin. FIBRINOLYSIS: DEGRADATION OF FIBRINOGEN & FIBRIN
• Due to competitor. it will not BY PLASMIN
have enough plasmin left
that can bind to the fibrin clot
that will cause fibrinolysis.

STRUCTURE OF FIBRINOGEN

Figure 9.3 Structure of Fibrinogen

• When the fibrinogen starts to polymerize with other molecules,
it connects the E domains and D domains to another set
of E domains and D domains.
❖ Fibrinogen is cleaved by Thrombin at Fibrinopeptides A Figure 9.5 Fibrinolysis: Degradation of Fibrinogen and Fibrin
and B. by Plasmin; Right (Secondary Hemostasis); Left (Primary
➢ Fibrinopeptide A: found in α alpha chain. Hemostasis)
➢ Fibrinopeptide B: found in β beta chain. • Plasmin cuts or cleave at different sites forming the
degradation products such as DED Complex, E, and D-
dimer.
 MECHANISM OF FIBRINOLYSIS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

o D-dimer is only found or produce from cleave fibrin Table 9.1 Difference Between Laboratory Results
polymer. Laboratory Primary
o D-Dimer Test: an important laboratory test that serves as DIC
an indicator for thrombosis. Tests Fibrinolysis
• In fibrinogen, plasmin can also act and cleave them into PT Prolonged Prolonged
different degradation products. APTT Prolonged Prolonged
1. In the first level of cleavage, it will remove the α chain
and β chains found in the E domain forming the Fibrinogen ↓ ↓
Fragment X. Platelets ↓ Normal
2. Another level of cleavage in plasmin will cleave
forming the Fragment Y which is made up of D and E FSP Present Present
Domain. D - Dimer Positive Negative
3. Finally, after another round of plasmin cleavage it will
divide it into Fragments D and E. RBC
Schistocytes Normal
Morphology
FIBRIN DEGRADATION PRODUCT
Fragments X, Y, D and E and D-D (D - dimer) Reasons for Table 9.1
• Produced by digestion of fibrin or fibrinogen by plasmin. • Fibrin Split Products, also known as Fibrin
o D-Dimer (D-D) Degradation Products
➢Formed from cross-linked fibrin only. • In both types of fibrinolysis, there is now depleted
➢One of the most common laboratory tests performed, supply of fibrinogen because they were all already
and it is known as the specific marker for cleaved by plasmin so there will be no clot formation,
thrombosis and fibrinolysis. thus, prolonged both PT/APTT, & decreased
➢It is used to identify chronic and acute fibrinogen assay.
Disseminated Intravascular Coagulation (DIC), • Platelets are decreased only in DIC because primary
which is also available to diagnose in PT, and APTT. fibrinolysis is only concerned with excessive amount of
❖ Known as 2 D domains from separate fibrin plasmin and plasminogen activator.
molecules cross-linked by the actions of Factor • D-Dimer is only positive in DIC because D-Dimer only
XIIIa. come from degraded fibrin clot.
• Inhibit Hemostasis o Primary fibrinolysis can only occur in the
o Prevents platelet activation. absence of fibrin formation.
o Hinders fibrin polymerization. o DIC undergoes the process of fibrin clot
➢The presence of these degradation products in the formation up to fibrinolysis.
circulation is like a feedback mechanism. FIBRINOLYSIS LABORATORY TESTS
➢Upon their appearance, there is further prevention
or inhibition of hemostasis or clot formation.
WHOLE BLOOD CLOT LYSIS TIME (WBCLT)
PATHOLOGIC FIBRINOLYSIS
WB will clot when collected in a
Principle
glass tube without anticoagulant
The clot should remain intact for
Normal Result
48 hours at 37oC
• Clot lysis prior to 48 hours or 2
Excessive days.
Systemic • If there is shortened lysis time,
Fibrinolysis it indicates that it is very active
fibrinolytic proteins.

EUGLOBULIN CLOT LYSIS TIME TEST

• An old test which is time honored approach to measurement
of fibrinolytic activity (increased or decreased).
• Euglobulin
o Are proteins that precipitate when plasma is diluted
PRIMARY FIBRINOLYSIS with water and acidified with 1% acetic acid (pH 5.35
• Excessive amounts of plasminogen activators from to 5.40).
damaged cells or malignant cells. o Contains plasminogen-plasmin and plasminogen
o Converts plasminogen to plasmin in the absence of activators that are involved in fibrinolysis.
fibrin formation.
Fibrinolysis Deficiencies Occur When
• Unmonitored circulation of free plasmin, that leads to fatal
hemorrhage. • TPA or Plasminogen levels become depleted
SECONDARY FIBRINOLYSIS • When excess secretion of PAI-1 depresses TPA
• Disseminated Intravascular Coagulation: it is an
uncontrolled, inappropriate formation of fibrin within the
blood vessels secondary to infection (Sepsis), Neoplasm
(Acute Promyelocytic Leukemia), snake bite.
 MECHANISM OF FIBRINOLYSIS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

Prolonged Fibrinogen
Low level of TPA or excess May decrease in 4 - 24 hours
Euglobulin Clot Levels
secretion of PAI-1
Lysis Time Decrease up to 48 hours after
Platelets
Time Required for onset
>2 hours (2 - 10 hours)
Complete Lysis Primary
Negative
Lysis < 2 hours Increased fibrinolytic activity Fibrinolysis

Associated Conditions
Procedure
1. Euglobulin is diluted with water and acidified with 1% • Acute and Chronic DIC
acetic acid (pH 5.35 to 5.40). • Systemic Fibrinolysis
2. Refrigerate for 30 minutes.
• Deep Vein Thrombosis
3. Centrifuge, decant, and precipitate is redissolved in
Borate buffer + Thrombin / Calcium Chloride to • Pulmonary Embolism
induce clot formation. • Stroke
a. Euglobulin Precipitate: Plasminogen, Plasmin,
• Thrombolytic Therapy
Fibrinogen and Plasminogen Activators (euglobulin
fractions) Quantitative D - Dimer
GEL FORMATION TESTS FOR FIBRINOLYSIS • Microlatex particles in buffered
saline are coated with monoclonal
Protamine Sulfate Test anti-D-dimer antibodies.
• Detects fibrin monomer polymerization through gel • The coated particles are
formation (positive result). Principle
agglutinated by patient plasma d -
• Utilizes protamine sulfate and 50% ethanol. dimer; the resultant turbidity is
• Used to assess primary fibrinolysis. measured thru turbidimetric or
nephelometric means.
Procedure
1. Add Protamine Sulfate or 50% Ethanol to Platelet Poor Detection
Plasma (PPP). Limit 10ng/mL
2. Observe for gel formation. (Sensitivity)

Qualitative D - Dimer
Ethanol Gelation Test
• Detects the presence of fibrin monomers in plasma. SimpliRed D - Dimer
• Distinguish primary and secondary fibrinolysis.
Take Note! It is expected to have a negative result in primary Manual method that uses
fibrinolysis because it can occur even in the absence of Definition monoclonal antibody-coated visible
fibrin formation. latex particles
PRIMARY VS. SECONDARY • Suited to low or near patient
applications.
Primary Fibrinolysis: No Clot Formation Advantages ❖ Bedside.
Fibrin Monomer None ❖ D-dimer is usually STAT due to
Fibrin Polymer None patient s/sx.
D - Dimer None Abnormally high level of Fibrin
Protamine Sulfate Test (+) D - Dimer Degradation Products in the body (clot
Negative
(PST) formation/lysis)
Ethanol Gelation Test
Negative FIBRIN DEGRADATION PRODUCTS IMMUNOASSAY
(EST)
PST & EST detects Fibrin Monomer; all are negative since these • Known as Qualitative agglutination immunoassay.
parameters require fibrin formation, & does not happen in • Thrombo - Wellcotest Kit: Polystyrene latex particles in
primary fibrinolysis buffered saline are coated with anti-D and E fragment-
specific polyclonal antibodies calibrated to detect FDPs at
D – DIMER 2 mg/mL or greater.
• Fragment that results from lysis of fibrin by plasma. • Used in diagnosis and monitoring DIC.
• Important marker for thrombosis and fibrinolysis
• Marker for Disseminated Intravascular Coagulation (DIC). Samples Used Serum or Urine
Disseminated Intravascular Coagulation Test & PLASMINOGEN CHROMOGENIC SUBSTRATE ASSAY
Results • Congenital plasminogen deficiencies are mostly
Positive as soon as 4hrs after associated with Thrombosis.
D - Dimer Test
onset
No Plasminogen = No degradation of Fibrin
 MECHANISM OF FIBRINOLYSIS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

• Acquired plasminogen deficiencies are seen in DIC and

Acute Promyelocytic Leukemia (M3). Supernatant PPP Frozen at -70oC
Plasma
Positive Production of color (Yellow)
concentration of Enzyme immunoassay
Thrombolytic therapy is ineffective TPA antigen
Significance
when plasminogen levels are low
• Depleted TPA or plasminogen Tissue Plasminogen Activator Activity
levels. • It activates plasminogen.
• The intensity of the color is
• Excess secretion of PAI-1 directly proportional to the
Hypofibrinolysis suppressing TPA activity. activity of the analyte.
• Excess TPA or Plasminogen
levels, leads to decreased PAI- Reagent Plasminogen
1 secretion. Plasma TPA activates plasminogen,
Mimics the natural enzymatic and the resultant plasmin activity is
Substrates
process Principle measured using a chromogenic
Reagent streptokinase activates substrate which will give a color at
plasminogen to form plasmin the end of the reaction.
Streptokinase: An exogenous 1.1
Principle Reference Interval Upper Limit
plasminogen activator derived units/mL
from cultured of beta hemolytic Upper Limit for TPA Antigen
14ng/mL
streptococci Concentration
5 - 13.5mg/dL Normal Plasminogen Activity
PLASMINOGEN ACTIVATOR INHIBITOR - 1 ASSAY
• Serum PAI-1 Deficiency: Confirmatory for PAI-1 deficiency.
Decreased (Thrombosis) Increased (Bleeding) • The Incidence of Complete PAI-1 Deficiency
o Elevated in the Old Order Amish population of eastern
Thrombolytic Therapy Systemic Fibrinolysis and southern Indiana implying a pathogenic founder
Disseminated variant.
Intravascular Coagulation Acute Inflammation • Specimen Collection
(DIC) o Blood is collected from patients at rest.
o Acidified Citrate Tube (Stabilyte) and centrifuged
Hepatitis Pregnancy immediately to make PPP (prevents the in vitro release
Cancer - of platelet PAI-1).
Hereditary Condition - 3 Methods
• Thrombolytic Therapy: Decreased blood clots used to help PAI-1 Antigen
in clot lysis. Immunogenic
Methods
• DIC: Uncontrolled production of clot = Plasminogen is used
PAI-1 activity
• Hepatitis: Plasminogen is produced in liver Chromogenic Substrate
Methods
• Cancer: Neoplasm can activate TPA
Enzyme
Method for Functional PAI - 1
Immunoassay

Enzyme Immunoassay
• Known as chromogenic substrates.
• The intensity of the color is inversely proportional to the
activity of PAI-1.
TISSUE PLASMINOGEN ACTIVATOR ASSAY • Highly intense color at the end of the reaction is an
• A very sensitive test. indicative of low PAI-1 activity.
Reagent Urokinase
Specimen Collection
• TPA Activity The urokinase-PAI-1 complex is
o Diurnal Variation: Take note of the time of the day. immobilized with solid-phase monoclonal
o Increases After Exercise: Exercise can affect or anti-PAI-1 and is measured using
Principle
cause false results of TPA. monoclonal antiurokinase
immunoglobulin as the detecting
• Patients should be at rest.
antibody.
• Minimal tourniquet application
• Record the collection time
• Immediate acidification using Biopool Stabilyte
Acidified Citrate Tube (DiaPaharma) to prevent
degradation or neuralization of TPA (more stable
sample).
 QUANTITATIVE PLATELET DISORDERS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

OUTLINE
I Introduction: Bleeding Disorders
II Thrombocytopenia
III Significant Platelet Levels
IV Types of Thrombocytopenia
V Decreased Platelet Production
A Congenital Types of Impaired Platelet Production
i May - Hegglin Anomaly THROMBOCYTOPENIA
ii Thrombocytopenia with Absent Radius (TAR)
Syndrome
• Most common cause of clinically important, and commonly
iii Fanconi Anemia encountered in dengue.
iv Congenital Amegakaryocytic Thrombocytopenia • Decreased platelet count (<100,000 platelets/uL), and
v Autosomal Dominant Thrombocytopenia increased platelet production.
vi X - Linked Thrombocytopenia o Platelet Reference Value: 150,000 to 450,000
B Neonatal Thrombocytopenia platelets/uL.
i TORCH Syndrome
VI Acquired Types of Impaired Platelet Manifestations
A Drug - Induced Hypoplasia
B Ineffective Thrombosis (Platelet Production) • Petechiae
C Infection • Purpura
D Bone Marrow Infiltration
VII Increased Platelet Destruction • Ecchymoses or Bruise
VIII Immune Mechanisms of Platelet Destruction
A Immune Thrombocytopenic Purpura (3CM) SIGNIFICANT PLATELET LEVELS
i Acute Immune Thrombocytopenic Purpura (AITP)
ii Chronic Immune Thrombocytopenic Purpura (CITP)
B Immunologic Drug - Induced Thrombocytopenia <100,000/uL Abnormally low
i Drug - Dependent Antibodies 30,000/uL - 50,000/uL Bleeding possible with trauma
ii Hapten - Induced Antibodies
iii Drug - Induced Autoantibodies Spontaneous bleeding
iv Immune Complex - Induced Thrombocytopenia <30,000/uL possible even without
C Neonatal Immune - Mediated Thrombocytopenia trauma
i Alloimmune Mechanism
ii Autoimmune Mechanism Severe, spontaneous
D Post Transfusion Purpura <5,000/uL
bleeding
E Secondary Thrombocytopenia, Presumed to be Immune -
Mediated TYPES OF THROMBOCYTOPENIA
IX Nonimmune Mechanisms of Platelet Destruction
A Thrombocytopenia in Pregnancy & Pre - Eclampsia • Due to decreased platelet production (bone marrow)
B Hemolytic Disease of the Newborn (HDN) • Due to decreased platelet destruction.
C Thrombotic Thrombocytopenic Purpura (TTP) • Due to an abnormal distribution or dilution (spleen or
D Hemolytic Uremic Syndrome (HUS) sequestration)
E Atypical Hemolytic Uremic Syndrome (AHUS)
F Disseminated Intravascular Coagulation (DIC) Table 10.1 Classification of Thrombocytopenia: Impaired or
G Purpura Fulminans/Devastating Thrombotic Disorder Decreased Production of Platelets
H Nonimmune Drug - Induced Thrombocytopenia
X Abnormalities in Distribution or Dilution Classification Thrombocytopenia
XI Thrombocytosis May - Hegglin Anomaly
XII Reactive Thrombocytosis/Secondary Thrombocytosis Bernard - Soulier Syndrome
A Process Resulting in Thrombocytosis
B Conditions that Lead to Secondary Thrombocytosis Fechtner Syndrome
C Kawasaki Disease Sebastian Syndrome
XIII Primary/Autonomous Thrombocytosis Epstein Syndrome
A Myeloproliferative Disorders
i Essential Thrombocytopenia Montreal Platelet Syndrome
ii Chronic Myelogenous Leukemia Fanconi Syndrome
Congenital
iii Polycythemia Vera Wiskott - Aldrich Syndrome
iv Myelofibrosis with Myeloid Metaplasia Thrombocytopenia with Absent Radius
(TAR) Syndrome
INTRODUCTION: BLEEDING DISORDERS Congenital Amegakaryocytic
• It causes platelet abnormalities, qualitative and Thrombocytopenia
quantitative due to bleeding disorders. Autosomal Dominant & X - Linked
Common Symptoms Thrombocytopenia
Viral or bacterial infections
• Petechiae: Small pinpoint 1mm Acquired
Drug induced
• Purpura: Small round leakage of blood 3mm Neonatal -
• Ecchymoses: Irregular, large leakage of blood 1cm
• Epistaxis: Nosebleed
• Gingival Bleeding: Mouth bleed
 QUANTITATIVE PLATELET DISORDERS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

DECREASED PLATELET PRODUCTION Absent Radius Lack of radial bone in arm

Autosomal Recessive
CONGENITAL TYPES OF IMPAIRED PLATELET Genetics
Disorder
PRODUCTION
RBM8A gene of chromosome
Mutations
May – Hegglin Anomaly 1
• Lack of adequate Bone Marrow (BM) megakaryocytes.
Some Cases Increased, abnormal structure Characteristics
• Enlarged platelets and misshapen • Severe neonatal thrombocytopenia.
PBS
platelets (>20um). • Congenital absence or extreme hypoplasia of the
• Dohle-like bodies in neutrophils radial bones of the forearms with absent, short, or
and monocytes. malformed ulnae and other orthopedic abnormalities.
Platelet • Cardiac Lesions.
Normal
Function • Transient leukemoid reactions with elevated WBC
• Mostly asymptomatic count (>100,000/uL).
Signs &
• Severe cases may require
Symptoms
transfusion Fanconi Anemia
Genetics Autosomal Dominant Disorders 21 genes start with FANC- followed by an
Mutation MYH9 Gene alphabet depending on chromosome being
affected.
• Sebastian Syndrome
Other Related • Autosomal Recessive Disorder
• Fechtner Syndrome Genetics
Diseases • Congenital bone marrow failure syndrome
• Epstein Syndrome

Characteristics
• Sebastian Syndrome
o It is an autosomal dominant disorder that contains • Aplastic anemia (Pancytopenia).
dohle - like bodies. • Abnormalities of bones and visceral organs.
o Characterized by large platelets, thrombocytopenia,
and granulocytic inclusions. Congenital Amegakaryocytic Thrombocytopenia
• Fechtner Syndrome • Autosomal Recessive Disorder causing bone marrow failure.
o Similar to Sebastian Syndrome, which focuses on kidney
and eyes. MPL gene of chromosome 1 leading to
o Accompanied by deafness, cataracts, and nephritis. Mutations complete loss of thrombopoietin
• Epstein Syndrome receptor function
o Characterized by large platelets and focuses on kidney
and eyes.
o It is mostly associated with deafness, ocular problems, Characteristics
and glomerulonephritis. • Low platelet count (<20,000/uL) at birth.
Thrombocytopenia with Absent Radius (TAR) Syndrome • Bleeding.
• Platelet count tends to rise and often normalizes in adulthood. • Physical Anomalies.

Table 10.2 Markers at Each Stage of Megakaryocyte Maturation Detected by Flow Cytometry, Immunostaining, Fluorescence In Situ
Hybridization, or Chemical Stain
BFU - CFU - LD - CFU - MK - MK -
Megakaryocyte/Platelet Membrane Marker MK - I PLT
Meg Meg Meg II III
MPL, TPO Receptor by FCM
CD34, Stem Cell Marker by FCM
CD41, αIIb Portion of αIIβ3, Peroxidase by
TEM Cytochemical Stain
CD42, GB Ib Portion of VWF Receptor by
FCM
PF4, by FCM
VWF, by Immunostaining
Fibrinogen, by Immunostaining
 QUANTITATIVE PLATELET DISORDERS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

Autosomal Dominant Thrombocytopenia

ANKRD26 gene of chromosome 10
Mutation Leads to incomplete megakaryocyte
differentiation
Platelets Size, shape, and function are normal
Signs &
Absent or mild bleeding
Symptoms

X - Linked Thrombocytopenia
Mutation WAS or GATA1 genes

Small Mild thrombocytopenia, absent or mild

Platelets bleeding
Large
Severe bleeding
Platelets

NEONATAL THROMBOCYTOPENIA
Platelet
<150, 000/uL
Count
ACQUIRED TYPES OF IMPAIRED PLATELET
75% of Thrombocytopenia is present at or
Cases within 72 hours of birth DRUG – INDUCED HYPOPLASIA
Definition A problem that occurs in maternal • Use of chemotherapeutic agents.
o Suppresses production of hematopoietic cells (including
megakaryocytes).
Causes of Neonatal Thrombocytopenia o Examples (no need to memorize): Methotrexate,
1. Toxoplasmosis, Other (Treponema pallidum, Varicella Busulfan, Cytosine, Arabinodies, Cyclophosphamide, and
Zoster Virus, Parvovirus B19), Rubella, Cisplatin.
Cytomegalovirus, Herpes. Drug - Induced
2. Maternal ingestion of sulfonamides (toxic drugs). Dose - limiting factor of treatment
Thrombocytopenia
a. Chlorothiazide Diuretics: patients with edema Treatment Recombinant interleukin-11
to expel excess water.
b. Tolbutamide Oral Hypoglycemic Medication: • Zidovudine: HIV treatment
Thrombocytosis • Anagrelide
patients with diabetes. Treatment
• Ethanol: not a drug but causes
TORCH Syndrome thrombocytopenia if long term
use.
Characteristics small platelets Other Drugs • Interferon Therapy
• Most common infectious agent • Estrogenic Drugs
causing congenital (Diethylstilbestrol)
Cytomegalovirus thrombocytopenia. • Antibiotics
(CMV) • Inhibits megakaryocytes and • Tranquilizers
their precursors, resulting to • Anticonvulsants
impaired platelet production

Take Note!
Congenital types are associated with mutations while
neonatal types are associated with the mother’s
condition

Figure 10.1 Mechanism of Drug - Induced Thrombocytopenia

 QUANTITATIVE PLATELET DISORDERS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

Gestational Thrombocytopenia [in

• Top Panel: Certain drugs stimulate the production of
pregnancy. preeclampsia, and
immunoglobulins that bind a platelet membrane
HELLP syndrome)
antigen or antigen and drug combination;
monocyte/macrophage Fc receptors bind the Fc portion Human Immunodeficiency Virus infection
of the immunoglobulin bound to the platelet resulting in Hemolytic Disease of the Newborn
Nonimmune
platelet removal. Thrombotic Microangiopathies (including
• Bottom Panel: Mechanisms of four specific drugs are TTP, HUS, aHUS)
depicted. GP, Glycoprotein; HIT, heparin-induced Disseminated Intravascular Coagulation
thrombocytopenia; IgG, immunoglobulin G; PF4, platelet Bacterial infection, other infections/sepsis
factor 4. Drug induced
Splenic sequestration (often associated
INEFFECTIVE THROMBOSIS (PLATELET PRODUCTION) with hypersplenism, chronic liver
• Characteristics of megaloblastic anemias, which usually disease)
Distribution
caused by impaired DNA synthesis (def. vitamin B12 or or Dilution
Kasabach-Merritt Syndrome
folic acid/folate). Hypothermia
• Considered also as qualitative disorder. Loss of Platelets: massive blood
Increased megakaryocytes but transfusions, extracorporeal circulation
“Ineffective”
decreased amount of platelets released IMMUNE MECHANISMS OF PLATELET DESTRUCTION
large platelets with abnormal function • Involves antibody formation and immune complexes.
PBS
and decreased survival time
Vitamin replacement (Folic Acid & Vitamin IMMUNE THROMBOCYTOPENIC PURPURA (ITP)
Treatment • Previously thought of as idiopathic (caused to be unknown).
B12)
• It has 2 types, which are acute and chronic.
INFECTION
Table 10.4 Difference Between Laboratory Results
CMV, varicella-zoster virus, rubella virus, Characteristics Acute ITP Chronic ITP
Viruses
Epstein-Barr virus, dengue virus Age at Onset 2 - 6 years old 20 - 50 years old
Neisseria meningitidis Sex Female over
Bacteria None
(meningococcemia) Predilection male (3:1)
BONE MARROW INFILTRATION Prior Infection Common Unusual
• Myeloma, lymphoma, metastatic cancer, and myelofibrosis. Onset of
• Equivalent to cancer. Sudden Gradual
Bleeding
Lack of space in Bone Marrow → No Space for Platelets 30,000 -
<20,000/uL
Megakaryocytes → No Space for Platelets Count 80,000/uL
INCREASED PLATELET DESTRUCTION Duration 2 - 6 weeks Months to years
• Caused by immunologic responses. Spontaneous
90% of patients Uncommon
• It is caused by mechanical damage, consumption, or Remission
sequestration. Higher incidence
Seasonal
Table 10.3 Classification of Thrombocytopenia: Increased Pattern
in winter & None
Platelet Destruction & Dequestration spring
Classification Associated Disorders 30% response
Acute and Chronic Immune rate.
Thrombocytopenic Purpura • <45yrs old,
Therapy: 70% response
Drug induced: immunologic 90% response
Steroids rate; rarely
Heparin-induced Thrombocytopenia rate
Splenectomy required
Neonatal Alloimmune Thrombocytopenia • >45yrs old,
Immune
Neonatal Autoimmune 40% response
Thrombocytopenia rate
Post Transfusion Purpura
Secondary Autoimmune
Acute immune thrombocytopenic purpura (AITP)
Thrombocytopenia • Disorder of children, that is caused by infection or
vaccinations (MMR, DTP, polio, Hepatitis A or B) producing
antibodies or immune complexes that bind to platelets.
Abrupt onset of bruising
petechiae,
Characteristics mucosal bleeding →
thrombocytopenia (1 -3 weeks
after infection)
Mild Scattered petechiae
 QUANTITATIVE PLATELET DISORDERS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

Extensive petechiae, ecchymoses, Hapten – Induced Antibodies

Common • Small drug molecules bind to large
hematuria, and/or epistaxis
plasma proteins or Platelet membrane
PLT (>10,000/uL), non - cutaneous proteins to induce antibody form action.
bleeding such as gastrointestinal • Bleeding is often severe and rapid in
Severe
bleeding, hematuria, mucous onset, and hemorrhagic bullae in the
membrane bleeding, and retinal mouth may be prominent.
hemorrhage, intracranial • Common Drugs: Penicillin and its derivatives
hemorrhage
Drug – Induced Autoantibodies
Recovery 3 weeks to 6 months • Drugs stimulate the formation of an autoantibody that binds to
Treatment Corticosteroids, Intravenous a specific platelet membrane glycoprotein with no
(assess Immunoglobulin (IVIG), Anti - D requirement for the presence of free drug.
bleeding) Immunoglobulin • Gold salts, Procainamide, Levodopa.
Immune Complex – Induced Thrombocytopenia
Chronic Immune Thrombocytopenic Purpura (CITP) • Also known as ‘Heparin - Induced Thrombocytopenia (HIT)’
• Commonly among patients 20 - 50 years old, and it is more • There is a complex of antibody and platelets that needs to be
common among women. formed for the body to lead to thrombocytopenia.
Onset Gradual Memorize all the drugs under ‘Immunologic Drug-Induced
Recovery >6 months Thrombocytopenia’ and their mechanisms.
Mucocutaneous bleeding, with
Sign &
menorrhagia, recurrent epistaxis, and
Symptoms
easy bruising (ecchymoses)
Cause Binding of autoantibodies to platelets
Corticosteroids, IVIG, anti-D
immunoglobulin, splenectomy,
Treatment
immunosuppressive drugs, platelets
transfusions

IMMUNOLOGIC DRUG – INDUCED THROMBOCYTOPENIA

• Due to development of drug dependent antibody after 1 - 2
weeks of exposure.
Identification of the offending drug,
drug discontinuation, and substitution
Treatment
with another suitable therapeutic
agent
1. Heparin binds to PF4 or PF4 binds to platelet membrane.
In Cases of a. PF4: it is a protein secreted by platelets (alpha
Platelet transfusion, high-dose IVIG
Bleeding granules)
2. Neoepitopes of PF4 are exposed.
Drug – Dependent Antibodies 3. The IgG Fab binds to neoepitope. IgG binds to platelet Fcγ IIa
• The fab region of the induced antibodies binds to the receptor.
GPIb/IX/V complex or the GPIIb/IIIa complex of the platelets 4. Platelet activation → aggregation → consumption
only in the presence of the drug.
• The Fc region will remain then bind to phagocytic cells. NEONATAL IMMUNE – MEDIATED THROMBOCYTOPENIA
• The main problem is through maternal, not the neonates.
Mostly Used Quinidine, Quinine, and
Drugs Sulfonamide Derivatives Alloimmune Mechanism
Induced
Mostly IgG
Antibodies
Drugs with
Abciximab, Eptifibatide, and Tirofiban
Similar
(antiplatelet agents)
Pattern
 QUANTITATIVE PLATELET DISORDERS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

1. Mother lacks platelet specific antigen (HPA -1a).

2. The baby inherited the said antigen from the father.
3. Antigen goes to the mother's circulation.
4. Mother’s antibody attacks the fetus.
Autoimmune Mechanism
• Passive transplacental transfer of antibodies from a
mother with ITP or occasionally, Systemic Lupus
Erythematosus.
POST TRANSFUSION PURPURA
• Develops 1 week after transfusion of products with
platelets.
• The recipient has alloantibody against the platelet antigen
transfused.
• Common Alloantibody: against HPA-1a antigen.
SECONDARY THROMBOCYTOPENIA, PRESUMED TO BE
IMMUNE – MEDIATED
• Seen in interferons, CSFs, Interleukin-2 therapies, SLE,
Malaria.
NONIMMUNE MECHANISMS OF PLATELET DESTRUCTION
• Thrombotic Microangiopathies: it is usually characterized
by thrombocytopenia, clot formation, within the arterioles HEMOLYTIC DISEASE OF THE NEWBORN (HDN)
and capillaries, and microangiopathic hemolytic anemia. • Due to the Rh incompatibility.
• RBC Destruction: antibody - mediated.
Caused By • Platelet Destruction: Due to the interaction with RBC
• Exposure of platelets to non-endothelial surfaces breakdown.
• Activation of the coagulation process
• Platelet consumption by endovascular injury

Most Common Conditions

• Thrombotic Thrombocytopenic Purpura
• Hemolytic Uremic Syndrome

THROMBOCYTOPENIA IN PREGNANCY & PRE –

ECLAMPSIA
• Also known as ‘Incidental Thrombocytopenia of
Pregnancy’, ‘Pregnancy Associated Thrombocytopenia’,
and the ‘Gestational Thrombocytopenia’.
• Pre - Eclampsia: it is a pregnancy characterized by
THROMBOTIC THROMBOCYTOPENIC PURPURA (TTP)
hypertension and can also have swelling and proteinuria. • It is referred to as ‘Moschcowitz Syndrome’.
• It is characterized by MAHA, thrombocytopenia, neurologic
Associated with hypertensive abnormalities, fever, renal dysfunction.
disorders (preeclampsia, • Most common in women 30 - 40 years old.
preeclampsia - eclampsia,
20% of Cases Presence of autoantibodies that attack
preeclampsia with chronic Cause
ADAMTS13
hypertension, chronic hypertension,
and gestational hypertension) Cleaves to von Willebrand Factor to
ADAMST13 prevent accumulation and
Microangiopathic Hemolysis,
HELLP spontaneous activation of the platelets
Elevated Liver Enzymes, and Low
Syndrome
Platelet Count
Treatment Delivery of infant 4 Types of TTP
• Single Acute Episode
• Recurrent
• Drug - Induced
• Chronic Relapsing (Upshaw - Shulman Syndrome):
occur at intervals, every 3 months, and it is an inherited
deficiency of ADAMST13.
 QUANTITATIVE PLATELET DISORDERS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

PURPURA FULMINANS / DEVASTATING THROMBOTIC

DISORDER
• Fulminant: means severe or deadly and escalates quickly.
• It starts with a purpura, but eventually will lead to tissue
destruction.
• Most common in neonates and children.
• Characterized by large irregular areas of blue - black
cutaneous bleeding that rapidly progress to necrosis of
superficial skin and deeper soft tissues.
Presenting of Acute Sepsis Caused by
• Neisseria meningitidis
• Streptococcus pneumoniae
• Group A and B streptococci
• Haemophilus influenza or Staphylococcus aureus (less
common)

HEMOLYTIC UREMIC SYNDROME (HUS) Widespread activation of the

• Side effect of bacterial infection caused by Shigella Mechanism
systemic inflammatory response, as
dysenteriae serotypes or enterohemorrhagic Escherichia (Leads to
coli OH serotypes, (0157:H7). well as the coagulation and
Tissue
• It resembles TTP but is more common in children and is complement pathways, which leads
Necrosis)
self - limiting. to increased bleeding
• Considered as HUS if no neurologic symptoms, the
presence of renal dysfunction, and the absence of other NONIMMUNE DRUG – INDUCED THROMBOCYTOPENIA
organ involvement. • No antibodies are involved, and directly activate the
• It is usually characterized by hemolytic anemia, renal failure, platelets.
and thrombocytopenia. • Ristocetin, Hematin, Protamine sulfate, Bleomycin.
ABNORMALITIES IN DISTRIBUTION OR DILUTION
• Platelets are present but hidden somewhere else primarily in
the spleen.
• “Big Spleen” (Splenomegaly) Syndrome: total platelet
mass is normal, but the majority is in the spleen instead of
the circulation.
Enlarged Spleen → ↑Sequestered PLTs → ↓PLT Count
Associated Diseases
• Hepatic cirrhosis
• Portal hypertension
• Hodgkin and non-Hodgkin lymphoma
• Sarcoidosis
• Leukemias

ATYPICAL HEMOLYTIC UREMIC SYNDROME (AHUS) • Gaucher disease

• Rare progressive disease that often has a genetic
component. Other Associated Conditions
• Not associated with infection, but with a defect in the
regulation of complement activation. • Hypothermia (distribution of platelets in the spleen)
↓Temp → Platelets becomes Sequestered → ↓PLT
DISSEMINATED INTRAVASCULAR COAGULATION (DIC) Count
• Thrombocytopenia + decreased clotting factors, and it is
similar to TTP. • After surgery (partial activation of platelets)
• Characterized by MAHA and deposition of thrombin in the • After transfusion of stored whole blood
arterial circulation of most organs.
o DIC: Platelets and fibrinogen (red clots) THROMBOCYTOSIS
o TTP: Platelets and vWF (white clots) • Secondary thrombocytosis because it is only a reaction to
Severe thrombocytopenia, life - another or underlying condition.
Acute DIC
threatening require immediate treatment • Reactive thrombocytosis
• Thrombocytosis Associated with Myeloproliferative
Chronic
Low - grade, not life - threatening, Disorders
treatment not urgent; caused by o Due to abnormal proliferation also called autonomous or
DIC
underlying condition primary thrombocytosis.
 QUANTITATIVE PLATELET DISORDERS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

KAWASAKI DISEASE SYMPTOMS

REACTIVE THROMBOCYTOSIS / SECONDARY
THROMBOCYTOSIS
• A non-pathological condition, wherein the platelet count are
450,000/uL and 800,000/uL with no changes in platelet
functions.
• Not associated with bleeding or thrombosis.
• Platelet levels return to normal soon as the underlying disorder
is brought under control (not as threatening as other types).
PROCESS RESULTING IN THROMBOCYTOSIS
• Reactive (Secondary)
o Moderate increase (Can reach 1,000,000/uL but rare).
o Asymptomatic
o Platelets: Responsive to Thrombopoietin (TPO) that
induces production of platelets
o Normal morphology
o Bone Marrow: normal to increased megakaryocytes with
normal morphology.
CONDITIONS THAT LEAD TO SECONDARY
THROMBOCYTOSIS
• Returns to normal after 10 -16
days.
Hemorrhage or • Associated with blood loss. PRIMARY / AUTONOMOUS THROMBOCYTOSIS
Surgery Thrombocytopenia → • A pathological condition that causes an uncontrolled
Thrombocytosis → Normal proliferation of platelets (which is known as characteristics
Count of Myeloproliferative disorders)
• Rebalancing of circulating platelet • Marked Elevation of Platelets
o Unregulated by TPO
Post pool. o Abnormal morphology and size
Splenectomy • May remain elevated for several
years. MYELOPROLIFERATIVE DISORDERS
• A persistent condition, that uses a clonal hematopoietic
• May reach 2 million/uL. disorder caused by genetic mutations in the hematopoietic
• Returns to normal within 7 days stems cells (HSCs) that result in expansion, excessive
Iron Deficiency
Anema
of iron therapy. production, and accumulation of mature erythrocytes,
• Iron (regulator): inhibits granulocytes, and platelets.
thrombopoiesis Essential Thrombocytopenia
• Thrombocytosis is indicative of • Chronic myeloproliferative neoplasm.
inflammation. • Most common cause of thrombocytosis.
• Kawasaki Disease • Diagnosed by excluding all causes of reactive thrombocytosis.
Thrombocytosis • Characterized by thrombocytosis greater than 1 million/uL
o Inflammation of walls of
associated with of platelet count and increased megakaryocytes.
arteries.
inflammation
o Acute febrile illness of infants Associated Diseases
and disease
and young children.
o Self-limited acute vasculitis • Thrombosis
syndrome of unknown origin. • Hemorrhage: occurs after thrombotic episode and it
• Release of platelets from splenic is related to platelet qualitative abnormalities.
Exercise - • Platelet Dysfunction: spontaneous aggregation of
pool
Induced
• Hemoconcentration. functionally abnormal platelets.
Thrombocytosis
• Returns after 30 minutes.
• Occurs after thrombocytopenia Treatment
caused by marrow-suppressive • Myelosuppressive agents
Rebound
drugs.
Thrombocytosis • Plateletpheresis
• Occurs after withdrawal from
offending drug. • Pegylated interferon
• Anagrelide
• Aspirin
• Hydroxyurea
• Ruxolitinib
 QUANTITATIVE PLATELET DISORDERS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

Chronic Myelogenous Leukemia

• It is a stem cell disorder affecting the granulocytic,
monocytic, erythrocytic, and megakaryocytic cells lines.
• Genetic mutation: translocation of one arm of chromosome
22 to chromosome 9 (Philadelphia chromosome).
• Patients who are negative for the Philadelphia
chromosome usually have a poorer prognosis and do not
respond particularly well to chemotherapy.
• It causes an increased M:E ratio in the Bone Marrow.
•
Polycythemia Vera
• It is an absolute increase in red blood cells, white blood
cells, and platelets.
• Malignant hyperplasia of the multipotential myeloid stem cells
causes an increase in all cell lines.
Myelofibrosis with Myeloid Metaplasia
• Fibrosis and granulocytic hyperplasia of the bone
marrow, with granulocytic and megakaryocytic in proliferation
in the liver and spleen.
 Chapter 11: Qualitative Platelet Disorders
Ms. Aila Nell Sarmiento | 2nd Semester | A.Y. 2023-2024

OUTLINE Platelet aggregating agents

I. INTRODUCTION
• Qualitative Platelet Disorders
✓ Characterized excessive bruising and
superficial (mucocutaneous) bleeding
even if the platelet count is normal
❖ Agonist serves as a panel in Immunoassay tests which
can help us understand the disease associated better.
B. Hereditary Afibrogenemia
• Platelet aggregation disorder similar to Glanzmann
thrombasthenia
• Characterized by hemorrhagic manifestations
• TREATMENT: Transfusion of Fibrinogen in
cryoprecipitate
II. DISORDERS OF PLATELET AGGREGATION • LAB RESULTS: (Fibrinogen-coagulation factor)
A. Glanzmann thrombasthenia ALL ARE PROLONGED/ABNORMAL
• Autosomal recessive disorder ✓ Platelet aggregation Test
• Due to lack of or a defect of (Glycoprotein) GP ✓ PT
IIb/IIIa (GP 2b/3a) in the platelet membrane ✓ APTT
❖ GP IIb/IIIa is the receptor for platelet aggregation, ✓ Reptilase
where platelets would be able to bind with other ✓ Thrombin
platelets and other procoagulants such as fibrinogen, ✓ Immunologic fibrinogen Assay
von willebrand factor, fibronectin III. DISORDERS OF PLATELET ADHESION
No GP IIb/IIa = No Aggregation = No Platelet plug A. Bernard-Soulier syndrome (BSS)
• Macrothrombocytopenia
REMEMBER: ✓ Platelets are large in size but small in volume
Process of Primary Hemostasis • Autosomal recessive
1. Adhesion • Deficiency of the GP Ib/IX/V complex on platelets
2. Aggregation ❖ GP Ib/IX/V complex is important for the binding of the
3. Secretion Von Willebrand factor (carpet-like) between the
END Product: Platelet plug endothelial cells and platelets
• Pseudo-BSS
• LAB RESULTS: (Platelet aggregation) ❖ has the presence of or production of antibodies to GP
✓ NORMAL: ristocetin Ib/V
✓ ABNORMAL (DECREASED): ECAT • Manifestation is SIMILAR/RESEMBLES Von
(Epinephrine, collagen, ADP, thrombin) Willebrand Disease
❖ Expect abnormal results or activity in the presence of ✓ the problem is in the Von Willebrand factor
different platelet agonist (aggregating agent) • LAB RESULTS:
✓ Platelet aggregation : ABNORMAL
✓ Using ristocetin: ABNORMAL (diminished
thrombin
✓ Using epinephrine, collagen, ADP :
NORMAL
• Treatment: apheresis platelets (transfusion)
 Chapter 11: Qualitative Platelet Disorders
Ms. Aila Nell Sarmiento | 2nd Semester | A.Y. 2023-2024

• Membrane defect present in hematopoietic cells

i. Montreal Platelet Syndrome (MPS) and lymphocytes
• Severe thrombocytopenia ✓ also associated with immune dysfunction
• Giant platelets such as Eczema-thrombocytopenia
• Characterized by abnormal and spontaneous platelet immunodeficiency syndrome
aggregation due to low calpain proteolytic activity ➢ meaning people with WAS have a high possibility of
✓ Calpain is an enzyme that cleaves binding being diagnosed with immune dysfunction.
proteins of platelets. No calpain means = • WASp: Role in actin cytoskeleton remodeling
No regulator of aggregation ✓ defect is the mutation of WAS protein =
• Bleeding Symptoms defect in the structural integrity of platelet
IV. DISORDERS OF PLATELET SECRETION • Characterized by small platelets and decreased
• Two types: dense granules
1. Storage pool disorders (deficiency/problems of B. Hermansky Pudlak
granules) • Dense Granule Defect
✓ Alpha granule defects • Autosomal recessive
a) Gray Platelet syndrome • Mutations in Chromosome 19
b) Quebec platelet disorder • SPECIAL TRAIT: Tyrosinase positive
c) Paris-Trousseau oculocutaneous albinism
✓ Dense granule defects ✓ Skin is white due to lack of melanin
a) Wiskott Aldrich syndrome ✓ Eyes are light colored sometimes clear
b) Hermansky Pudlak • Defective lysosomal Function
c) Chediak Higashi • Ceroid like deposition in the cells of the RES
• Profound dense granule deficiency
2. Release reaction defects (deficiency/problems of • Clinical Manifestation: It is deadly that may lead to
secretion of granules) lethal hemorrhage
A. Gray Platelet Syndrome C. Chediak Higashi
• Autosomal recessive disorder • Autosomal recessive
• Mutation in region 3p21 involving the gene • mutated protein gene on Chromosome 13
NBEAL2 ✓ leads to generalized cellular dysfunction
• Characterized by the specific absence of involving fusion of cytoplasmic granules
morphologically recognizable “alpha-granules” in • Characterized by:
platelets 1) Partial oculocutaneous albinism
✓ Platelets normally look like in a wright stain 2) Increased susceptibility to infections
film are purplish small with granules. 3) Giant lysosomal granules
Whereasin Gray platelet syndrome, the 4) Platelet dense granule deficiency
platelets look like a GHOST cell due to its 5) Hemorrhage
pale/gray appearance and absence of V. DISORDERS OF RECEPTORS AND SIGNALING
granules. PATHWAYS
• Peripheral Blood Smear: LARGE gray platelets on a A. ADP Receptors
Wright-stained blood film ❖ Remember that in platelets ultrastructure it has
• Clinical Manifestation: lifelong bleeding tendencies receptors which where many proteins bind to, to
B. Quebec Platelet Disorder facilitate hemostasis.
• Alpha Granule Defect ❖ Some of those receptors are attracted or bind to
• Autosomal dominant bleeding disorder agonists
• Characterized by: ❖ These receptors that if deficient hemostasis can’t
✓ Deficiency of multimerin stored in alpha- continue
granules leading to the degradation of many ✓ P2X1 - responsible for calcium ion influx
alpha-granule proteins ✓ P2Y1 - responsible for calcium mobilization
C. Paris-Trousseau and shape change in response to ADP
• Alpha Granule Defect ✓ P2Y12 - needed for platelet aggregation
• Congenital deficiency of the distal portion of • Deficiency: decreased platelet aggregation in
chromosome 11 response to ADP but normal platelet shape change
• Characterized by psychomotor retardation, facial and and calcium mobilization
cardiac abnormalities B. Scott Syndrome
• Enlarge platelet • Rare autosomal recessive disorder
• Giant Alpha granules • Characterized by the inability of platelets to “flip”
A. Wiskott Aldrich Syndrome their membrane lipids for coagulation factor
• Dense Granule Defect assembly
• SPECIAL TRAIT = Triad of: thrombocytopenia, ❖ the phospholipids of platelets in order to be activated
recurrent of infection and eczema. they need to be rearrange, if they do not change their
• X-linked disease position hinder hemostasis
• • Enzyme related is scramblase enzyme
 Chapter 11: Qualitative Platelet Disorders
Ms. Aila Nell Sarmiento | 2nd Semester | A.Y. 2023-2024

C. Stormorken Syndrome C. Multiple myeloma and Waldenstrom macroglobulinemia

• Opposite of Scott Syndrome • Multiple myeloma - Cancer of plasma cells and
• Platelets are always an “activated” state bone marrow
• Over expression of phosphatidylserine on the outer • Waldenstrom macroglobulinemia - low-grade
leaflet of the membrane without prior activation lymphoplasmacytic lymphoma
✓ nag sasariling mag activate or nagfliflip • Platelet function is impaired due to coating of the
• Enzyme defective: aminophospholipid translocase platelet membrane by paraproteins
VI. DRUG-INDUCED QUALITATIVE PLATELET DEFECTS • Fibrin polymerization and coagulation proteins
A. Drugs that inhibit the prostaglandin pathway may also be affected
• Aspirin D. Hyperaggregable platelets
- inhibits cyclooxygenase in the pathway, preventing • Associated with hyperlipidemia, diabetes mellitus,
the synthesis of TXA2 myocardial infarction, stroke, among others:
B. Drugs that inhibit membrane function ✓ leads to increased platelet reactivity, where
• P2Y12 (ADP) receptor inhibitors, GP IIb/IIIa (GP they react to aggregating agents at lower
2b/3a) receptor inhibitors, PAR-1 antagonists concentrations.
✓ All inhibit platelet aggregation 1) Sticky platelet syndrome
VII. DISORDERS THAT AFFECT PLATELET FUNCTION ✓ Autosomal dominant
A. Liver Disease ✓ Hyperaggregable platelets in response to
• Associated with a variety of hemostatic ADP, epinephrine, or both
abnormalities 2) Spontaneous aggregation
• Moderate to severe Liver disease: ✓ Aggregation in response to in vitro stirring
1) reduction in clotting proteins only
2) reduction of proteins in the natural anticoagulant VIII. Acquired storage pool Disease
pathways • Seen in patients with autoimmune disorders and
3) dysfibrinogenemia immune thrombocytopenias
4) excessive fibrinolysis • Characterized by excessive in vivo release of dense
• Chronic Liver Disease: granule contents caused by stimulation of thrombin
1) Mild to moderate thrombocytopenia and other agonist
2) abnormal platelet function test • Other name: Exhausted platelets
• Chronic Alcoholic cirrhosis:
✓ thrombocytopenia and platelet
abnormalities are caused by the direct
toxic effects of alcohol on Bone marrow
megakaryocyte
❖ Ethanol/Alcohol can affect platelet production
• End-stage liver disease: Severe bleeding diathesis
caused by:
✓ markedly decreased or negligible
coagulation factor production
✓ excessive fibrinolysis and
dysfibrinogenemia
✓ thrombocytopenia due to acquired
thrombopoietin deficiency
✓ splenic sequestration secondary to portal
hypertension
✓ Disseminated intravascular coagulation
B. Chronic renal failure (uremia)
• Kidney Disease = effect in platelet function
✓ there is an accumulation of waste product that
coats platelets
❖ Accumulation of Urea = uremia
❖ Accumulation of by products of the Urea cycle:
guanidinosuccinic acid (GSA) or dialyzable phenolic
compounds
❖ if platelets are coated they can’t perform their function
• Platelet activation, adhesion and aggregation are
suppressed because of platelet coating by
guanidinosuccinic acid or dialyzable phenolic
compounds
• Reversible via hemodialysis or renal transplantation
Chapter 11: Qualitative Platelet Disorders
Ms. Aila Nell Sarmiento | 2nd Semester | A.Y. 2023-2024
 CLASSIFICATION OF LEUKEMIA
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

OUTLINE Table 14.1 General Characteristics of Acute & Chronic

I Introduction
Leukemia
II Leukemias Chronic
A Etiology
Acute Leukemia
Leukemia
i Radiation: 3 Principal Lines of Study
ii Genetics Predominant
Precursor Cell or Blast Mature
iii Chemicals “Leukemogens” Cell Type
iv Viruses Onset Sudden Insidious
III Leukemoid Reaction
A Leukocyte (Neutrophil) Alkaline Phosphatase Test • Fever (as a result
i Kaplow’s Method of LAP of neutropenia-
IV Leukemia induced infection)
Variable,
A Classifications of Leukemia • Mucocutaneous
i According to Duration Symptoms & nonspecific;
bleeding (as a
ii According to Predominant Cell Type Presentation some
result of
iii According to WBC Count/cu.mm. in PBS asymptomatic
thrombocytopenia)
iv According to Age
B French – American – British (FAB) Classification of • Fatigue (as a
Leukemia result of anemia)
i Lymphocytic Leukemias White Blood
ii Non – Lymphocytic Leukemia Variable Increased
Cell Count
C WHO Classification of AML
V Cytochemical Stains of Leukemia Progression Slower,
Rapid; weeks to
VI Cell Markers Without months to
months
VII Myeloproliferative Neoplasms Treatment years
A Chronic Myelogenous Leukemia (CML)
B Essential Thrombocytopenia ETIOLOGY
C Polycythemia Vera
i Secondary Polycythemia Radiation: 3 Principal Lines of Study
ii Relative (Pseudo-) Polycythemia
D Primary Myelofibrosis • Radiologists who were occupationally exposed to
VIII Myelodysplastic Syndromes (MDSs) radiation before the establishment of safety standards and
A Classification of MDS practices in clinical radiology.
i MDS With Single Lineage Dysplasia (MDS-SLD) • Patients treated by radiation for ankylosing spondylitis
ii MDS With Multilineage Dysplasia (MDS-MLD) (as compared to those with same disease who were not
iii MDS With Ring Sideroblasts (MDS-RS) irradiated).
iv MDS with Excess Blasts (MDS-EB)
v MDS With Isolated Del (5q) (5q-Syndrome)
• Survivors of the atomic blasts at Hiroshima & Nagasaki.
vi MDS Unclassifiable (MDS-U) Genetics
vii Childhood Myelodysplastic Syndrome
• Down Syndrome (Trisomy 21)
IX Abnormal Cellular Function
• Fanconi Syndrome
• Bloom Syndrome
INTRODUCTION • D - Trisomy
• Hematologic Neoplasms: it is an abnormal growth of cells
of the hematopoietic system. Chemicals “Leukemogens”
o Leukemias: originate in Bone Marrow & proliferate in • Chloramphenicol
the peripheral blood. • Phenylbutazone
o Lymphomas: originate in the lymphatic system & • Arsenic – containing compounds
proliferate in lymph nodes and other lymphoid organs. • Sulfonamides
o Myelodysplastic Syndromes: progressive cytopenia • Some insecticides
in the peripheral blood & previously called • Phenylalanine mustard
“preleukemias”. • Cyclophosphamide
LEUKEMIAS • Alkylating agents
• Divided into lymphoid and myeloid lineages, acute & chronic. • Some immunosuppressants
• Acute Leukemia • Benzene: the only chemical known clearly to produce
o Sudden onset, rapid progression, fatal outcome in cancer.
weeks or months. Viruses
o Accumulation of precursor hematopoietic cells. • HTLV – 1
• Chronic Leukemia • Epstein – Barr Virus
o Gradual onset, slower progression, and longer survival. • HIV - 1
o Accumulation of mature and maturing cells.
• Leukemias can be developed by acquired genetic mutations LEUKEMOID REACTION
or environmental toxins. • Excessive leukocytic response in the peripheral blood.
• Leukocytosis of 50 x 109/L or higher.
• Does not signify a disease.
 CLASSIFICATION OF LEUKEMIA
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

• Not related to leukemia.

Decreased LAP Score
• Blood Picture Resembles Leukemia: increased white
blood cell (WBC) count with shift to the left. • CGL (Chronic Granulocytic Leukemia)
• Paroxysmal Nocturnal Hemoglobinuria
Due to
• Sideroblastic Anemia
• Infections
• Sickle Cell Anemia
• Inflammations
• Intoxications
Increased LAP Score
• Tumors
• Pregnancy
LEUKOCYTE (NEUTROPHIL) ALKALINE PHOSPHATASE TEST • Infections
• Differentiation of leukemoid reaction from leukemia. • Polycythemia Vera
• Principle: increased LAP activity can be observed in
neutrophils that have undergone normal growth. • Inflammations
• Also known as LAP Test. • Intoxications
• Tumors
Leukemoid Normal growth of neutrophils
Reaction (increased LAP score)
Malignant growth of neutrophils Procedure
Leukemia
(decreased LAP score) 1. Count 100 neutrophils and score each counted
neutrophil.
Kaplow’s Method Of Lap
a. 0 – No red/brown to black ppt.
• A smear is made from a drop of capillary blood (not EDTA
because it is inhibitory) then stained immediately. b. 1 – Slightly diffused red/brown to black ppt.
• Alkaline phosphatase in WBCs hydrolyzes naphthyl AS-B1 c. 2 – Moderately diffused red/brown to black ppt.
of the LAP stain. d. 3 – Heavily diffused red/brown to black ppt.
• 100 segmented neutrophils and bands are scored from 0 to e. 4 – Very heavily diffused red/brown to black
4+ based on stain intensity. ppt.
Normal • Example:
Kaplow’s 15 – 100 o 1st Neutro = 4
Score
o 2nd Neutro = 2
Red, brown to black precipitate of o 3rd Neutro = 0
Result alkaline phosphatase activity in the o So on and so forth
cytoplasm of neutrophils o 100th Neutro = 1
No. of Neutro X Score LAP Score
32 0 (32 x 0) = 0
24 1 (24 x 1) = 24
21 2 (21 x 2) = 42
15 3 (15 x 3) = 45
8 4 (8 x 4) = 32

LEUKEMIA
• It is a malignant growth of WBC – producing cells, RBCs,
and platelets.
More blasts → shorter, more fatal
General Rule
course of disease
Increased WBC count with shift to
Blood Picture
the left
M:E Ratio 10:1
Normal M:E
2:1 to 4:1
Ratio
Anemia Present
in Patient w/ Normocytic, Normochromic
Acute Leukemia
 CLASSIFICATION OF LEUKEMIA
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

CLASSIFICATIONS OF LEUKEMIA Types

According to Duration • Lymphocytic Leukemias (Acute Myeloid or AML)
• Non – Lymphocytic Leukemias (Acute
Acute High % of young forms Lymphoblastic or ALL)
Sub - Acute Slightly differentiated
Table 14.2 Acute Leukemia: Morphological Classification
Chronic High % of old forms
Acute Lymphoblastic
Acute Myeloid (AML)
According to Predominant Cell Type (ALL)
• Monocytic Leukemia Mo: minimally
L1: small, monomorphic
o Schilling’s Type: True monocytic leukemia differentiated
o Naegeli Type: May resemble granulocytic leukemia M1: without maturation L2: large, heterogenous
• Lymphocytic Leukemia
M2: with maturation L3: Burkitt-cell type
• Myelogenous Leukemia
o Philadelphia Chromosome: It is a translocation of the M3: hypergranular
long arm of chromosome 22 to the long arm of promyelocytic
chromosome 9 (9q+,22q-). M4: myelomonocytic
• Erythremic Myelosis M5: (a) monoblastic; (b)
o Pure erythroblasts monocytic
o Di Guglielmo’s Disease
M6: erythroleukemia
• Erythroleukemia
o Erythroblasts & myeloblasts M7: megakaryoblastic
o Di Guglielmo’s Syndrome Rare Types (e.g.,
• Mast Cell Leukemia eosinophilic, natural killer)
o Increased tissue basophils
o Stain Used: toluidine Blue (colored reddish violet) Lymphocytic Leukemias
• Plasma Cell Leukemia • All lymphocytic leukemias are negative for the
o Increased plasma cells Myeloperoxidase (MPO) stain and Sudan Black (SB) stain.
o Terminal stage of multiple myeloma MPO Primary granules
• Myelolymphocytic Leukemia
SB Lipids
o Increased granulocytes and lymphocytes
• Hairy Cell Leukemia
o “Hairy Cells”: lymphocytes with hair-like cytoplasmic
projections. • Most common type of leukemia in children.
Table 14.3 Types of ALL
According to WBC Count / CU.MM. in PBS
• Leukemic Leukemia L1 L2 L3
o High % of young forms. Histochem
o WBC Count: >15,000/cu.mm. PAS + + -
• Sub-Leukemic Leukemia
ORO - - +
o Slightly differentiated cells
o WBC Count: <15,000/cu.mm. MPO - - -
• Aleukemic Leukemia SB - - -
o Hard to diagnose Blasts
o High % of old forms
o WBC Count: <15,000/cu.mm. Burkitt Type:
Few
large
According To Age Many (small) (large &
lymphoblasts with
small)
vacuoles
Acute Leukemia Below 20 years
Chronic Lymphocytic Non - Lymphocytic Leukemias
Above 50 years old • Also known as “Myelogenous Leukemia”.
Leukemia
Chronic Granulocytic Between 20 and 50 years • All NLL are positive for MPO & SB.
Leukemia old

FRENCH – AMERICAN – BRITISH (FAB) CLASSIFICATION OF • Most common type in the elderly.
LEUKEMIA –
• Based on morphology of cells in Romanowsky-stained
smear. • Also known as “Acute Myelogenous Leukemia (AML)”.
• Based on cytologic and histochemical characteristics of cells • Types: M0 to M7
involved.
 CLASSIFICATION OF LEUKEMIA
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

M0 Myeloblastic with minimal differentiation CELL MARKERS

• Myeloid Antigens: CD 33 & CD 13
M1 Myeloblastic without maturation
• B Lymphoid Antigens: CD 19, CD 10, & CD 22
• Myeloblastic with maturation • T-Lymphoid Antigens: CD 2, CD 3, CD 5, CD 7
M2
• Most common type of AML • HLA-DR: present on both myeloid & B lymphoid cells.
• Promyelocytic, hypergranular MYELOPROLIFERATIVE NEOPLASMS
• Associated with faggot cells & DIC • Group of acquired malignant disorders that develop from the
M3
(disseminated intravascular coagulation) proliferation of abnormal pluripotent stem cells.
• M3V: Promyelocytic, microgranular • Clonal hematopoietic disorders caused by genetic mutations
• Myelomonocytic in the hematopoietic stem cells (HSCs) that result in
M4
• M4E: myelomonocytic w/ eosinophilia expansion, excessive production, and accumulation of
• Monocytic mature erythrocytes, granulocytes, and platelets.
M5 • M5a: monocytic, poorly differentiated • Characterized by one cell line only.
• M5b: monocytic, differentiated • May progress to acute leukemia.
M6 Erythroleukemia CHRONIC MYELOGENOUS LEUKEMIA (CML)
M7 Megakaryoblastic • Patients with this disorder who are negative for
Philadelphia chromosome have pooper prognosis and do
WHO CLASSIFICATION OF AML not respond well to chemotherapy.
• AML with recurrent cytogenic abnormalities • Decreased LAP score.
• AML with multilineage dysplasia • Philadelphia chromosome in all patients.
• AML therapy related Found Mainly Adults (45 years of age & older)
• AML not otherwise categorized
Weight loss, splenomegaly,
Symptoms
CYTOCHEMICAL STAINS FOR LEUKEMIA fever, night sweats and malaise
• Myeloperoxidase Bone Marrow Increased M:E ratio
o Enzyme found in primary granules of granulocytic Laboratory WBC between 50 and 500 x 106
cells (and to some extent monocytes).
o A positive peroxidase stain rules out ALL. ESSENTIAL THROMBOCYTOPENIA
• Sudan Black Stain • Characterized by proliferation of megakaryocytes.
o Stains the lipids in the granules of neutrophils & • Found primarily in adults 60 years of age and older.
monocytes.
o Reactions parallel those of the peroxidase stain. Platelet count greater than 1000
Laboratory
• Oil Red O cu.mm. (Giant forms)
o Stain for lipids, vacuoles in ALL.
• Periodic Acid Schiff Stain POLYCYTHEMIA VERA
o Stains lymphoblasts. • Malignant hyperplasia of the multipotential myeloid stem cell
• Esterase causes increase in all cell lines.
o Differentiates myeloblasts & neutrophilic granulocytes • Erythrocytes are mostly affected despite decreased EPO.
from cells of monocytic origin.
o Naphthol AS-D Chloroacetate: (+) granulocytes; (-) Found in Adults (50 years of age & older)
monocytes Panmyelosis
Increased BM production of RBC,
o a-Naphthyl Acetate: (+) monocytes; (-) granulocytes WBC, platelets
o a-Naphthyl Butyrate: diffuse positive in monocytes Pancytosis Peripheral blood smear
Table 14.4 Acute Leukemia Cytochemical Reaction Chart Polycythemia Increased hematocrit
MP SB NASD
Condition ANBE ANAE Secondary Polycythemia
O B A
• Caused By: smoking, emphysema & high altitude
-/+ -/+ • Increase in RBC mass in response to increased EPO or
ALL - - -
(focal) (focal) hypoxia.
AML + + + - - • Plasma volume, WBC count and platelet count are normal.
+ Relative (Pseudo-) Polycythemia
+
AMML + + + (diffus • Decreased plasma volume with normal RBC mass caused
(diffuse)
e) by dehydration (diarrhea, diuretics, or burns).
+
+ • Increased hemoglobin, normal leukocyte and platelet count,
AMoL - -/+ - (diffus normal EPO.
(diffuse)
e) • Increased Hct with normal RBC mass.
+
Megakaryoc
- - - - (localiz
ytic Anemia
ed)
 CLASSIFICATION OF LEUKEMIA
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

• Because of decreased plasma The 2016 World Health Organization Classification of

Increased volume. Myelodysplastic Syndromes (MDSs)
Hct • In dehydration • MDS with Single Lineage Dysplasia (MDS-SLD)
• Associated with stress & anxiety • MDS with Ring Sideroblasts (MDS-RS)
o MDS-RS & single lineage dysplasia
PRIMARY MYELOFRIBROSIS o MDS-RS & multilineage dysplasia
• Characterized by bone marrow hypercellularity,
extramedullary hematopoiesis, fibrosis, and increased • MDS with Multilineage Dysplasia
megakaryocytes. • MDS with Excess Blasts (MDS-EB)
• It is the least common but most aggressive form of MPN. o MDS-EB-1
o MDS-EB-2
MYELODYSPLASTIC SYNDROMES (MDSs)
• Myelodysplastic Syndrome with Isolated del (5q)
• Group of acquired clonal disorders affecting the pluripotent
stem cell. • Myelodysplastic Syndrome, Unclassifiable
• Characterized by progressive blood cytopenia despite BM • Childhood Myelodysplastic Syndrome
hyperplasia. o Refractory Cytopenia of Childhood (provisional)
• Defects in the erythroid, myeloid, and/or megakaryocytic
maturation. Classification of Myelodysplastic Syndromes or
• Can be triggered by chemotherapy, radiation, and chemicals Myeloproliferative Neoplasms
or viral infection.
• May transform to acute myeloid leukemia. • Chronic Myelomonocytic Leukemia (CMML)
• Found in older adults, rarely in children & young adults. • Atypical Chronic Myeloid Leukemia (aCML),
BCR/ABL1 negative
Table 14.5 Morphologic Evidence of Dyserythropoiesis,
• Juvenile Myelomonocytic Leukemia (JMML)
Dysmyelopoiesis, & Dysmegakaryopoiesis
• MDS/MPN with ring sideroblasts and
Dyserythrop Dysmyelopo
Dysmegakaryopoiesis thrombocytosis (MDS/MPN-RS-T)
oiesis iesis
• Myelodysplastic/myeloproliferative neoplasm,
Persistent
Oval unclassifiable
basophilic Giant platelets
macrocytes
cytoplasm MDS With Single Lineage Dysplasia (MDS-SLD)
Hypochromic Abnormal Platelets with abnormal • Dysplasia in at least one myeloid lineage.
microcytes granulation granulation • Characterized by cytopenia (anemia or increased infection
Dimorphic Abnormal or bleeding).
Circulating • BM Blasts <5% and peripheral blasts <1%.
RBC nuclear
micromegakaryocytes
population shapes MDS With Multilineage Dysplasia (MDS-MLD)
RBC • 1 or more cytopenia.
precursors Uneven • Dysplasia in 2 or more myeloid cell lines.
Large Mononuclear
with abnormal cytoplasmic • Blasts <1% in peripheral blood, <5% in bone marrow.
Megakaryocytes
nuclear staining • No Auer rods.
shapes
RBC MDS With Ring Sideroblasts (MDS-RS)
precursors Micromegakaryocytes or • SF3BA mutation.
with uneven micromegakaryoblasts • Ring Sideroblast: RBC precursor with at least 5 iron
cytoplasmic or both granules, circling at least 1/3 of nucleus.
staining • Characterized by anemia, dyserythropoiesis, and dimorphic
RBC.
Abnormal nuclear
Ring shapes in the
sideroblasts megakaryocytes/megaka
ryoblasts

CLASSIFICATION OF MDS
FAB
• Refractory anemia
• Refractory anemia with ring sideroblasts (RARS)
• Refractory anemia with excess blasts (RAEB) MDS With Excess Blasts (MDS-EB)
• Chronic myelomonocytic leukemia • Trilineage cytopenia.
• Refractory anemia with excess blasts in • Significant dysmyelopoiesis and dysmegakaryopoiesis.
transformation (RAEB-t) • MDS-EB 1: 5 – 9% blasts in bone marrow and 2 – 4% blasts
in peripheral blood.
 CLASSIFICATION OF LEUKEMIA
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

• MDS-EB2
o 10 – 19% blasts in bone marrow and 5 – 19% blasts in
peripheral blood.
o With Auer bodies.

MDS With Isolated Del (5q) (5q - Syndrome)

• Anemia without other cytopenia or thrombocytosis.
• Hypolobulated megakaryocytes.
• Erythroid hypoplasia.
• <1% blasts in peripheral blood.
MDS Unclassifiable (MDS-U)
• Reclassified once specific characteristics develop.
Childhood Myelodysplastic Syndrome
• Very rare.
ABNORMAL CELLULAR FUNCTION
• Shortened survival
RBC
• Decreased EPO response
• Decreased adhesion
• Deficient phagocytosis
WBC
• Decreased chemotaxis
• Impaired microbicidal capacity
Platelets Bleeding
 MATURE LYMPHOID NEOPLASMS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

OUTLINE
I Introduction
A Leukemia vs. Lymphoma
B Lymphatic System
II Lymphoma
III Lymphoma Classification
A WHO Classification
B Plasma Cell Neoplasms
i Diffuse Large B-cell Lymphoma
ii Follicular Lymphoma
C Hodgkin’s Lymphoma
i Subtypes (Rye Classification)
D Non-Hodgkin’s Lymphoma
i Classification of NHL (WHO/REAL)
E Burkitt’s Lymphoma
IV Hairy Cell Leukemia
A Key Differentiating Features
B Hairy Cell
C Clinical Features of Hairy Cell Leukemia

INTRODUCTION
• The strongest risk factor is altered immune function:
o Immunocompromised patients
o Persons with autoimmune disease
o Viral and bacterial infections
o Exposure to chemical and herbicides LYMPHOMA
LEUKEMIA VS. LYMPHOMA
Lymphadenopathy: refers to the
❖ Remember! Symptom
swelling of lymph nodes
➢ When neoplastic cells involve mainly the bone marrow
and blood, the disorder is known as leukemia. • CD Surface Markers
➢ When this disease is limited mainly to the lymph Diagnosis • Cytogenetics
nodes or organs, the disease is known as lymphoma. • DNA analysis/PCR
➢ Chronic Lymphocytic Leukemia
▪ An example of both leukemia and lymphoma
shares the same characteristic.
▪ There is a proliferation of lymphocytes & can also
involve the spleen in the lymph nodes.
Solid, malignant tumors of the
Lymphomas lymph nodes & associated
tissues
Lymphocytes & natural killer
Distinct Cell Type
cells

LYMPHATIC SYSTEM
• Consists of lymphatic lymph nodes and other lymphatic
organs.

LYMPHOMA CLASSIFICATION
• Revised European-American Lymphoma Classification
(REAL Classification).
• World Health Organization (WHO) Classification of Mature
Lymphoid Neoplasms.
REAL Classification
• Precursor B-cell Neoplasm
• Mature B-cell Neoplasm
• Precursor T-cell Neoplasm
• Mature T cell & NK cell Neoplasms
• Hodgkin Lymphoma
 MATURE LYMPHOID NEOPLASMS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

WHO CLASSIFICATION Diffuse Large B-Cell Lymphoma

• Mature B Cell Neoplasms • Characterized by diffuse tissue infiltration by large B-
o Plasma Cell Neoplasms lineage.
o Diffuse Large B Cell Lymphoma (DLBCL) • B Cell Prolymphocytic Lymphoma
o Follicular Lymphoma o It is characterized by medium-sized lymphoid cells with
• Mature T & NK Neoplasms distinct “punched out” nucleoli and abundant
• Hodgkin’s Lymphoma cytoplasm.
o 30% of cases • Mantle Cell Lymphoma
o Has a persistent defect in the cellular immunity with o It is characterized by medium-sized lymphoid cells with
abnormalities in T lymphocytes, IL-2 production. irregular nuclear outlines derived from the follicular
o Contains Reed-Sternberg Cells. mantle zone.
• Non-Hodgkin’s Lymphoma
Follicular Lymphoma
o 70% of cases.
• Also known as “Follicle Centre Cell Lymphoma.”
o Group of neoplastic disorders and include all
lymphomas except Hodgkin’s disease. • Lymphoma that originates from the germinal centers.
• Posttransplant Lymphoproliferative Disorders (PTLD) • Spleen is enlarged.
• B symptoms (fever, weight loss, & night sweats).
PLASMA CELL NEOPLASMS • Somewhat higher incidence in women.
Table 14.6 Mature B Cell Neoplasm
Diffuse Large B
Plasma Cell Cell Follicular
Neoplasms Lymphoma Lymphoma
(DLBCL)
B cell
Multiple myeloma prolymphocytic
lymphoma
Waldenstrom Mantle cell Figure 14.1 Clinical Photograph of a Patient with a Cutaneous
macroglobulinemia lymphoma Follicular Lymphoma

• It is a bone marrow-
based disease with
extramedullary
extension. Figure 14.2 Follicular Lymphoma Showing a Large Lymphoma
• Excessive IgG or IgA. Cell with a Deeply Cleft Nucleus with Giant Nucleoli
• Found in adults over 60
years old. HODGKIN’S LYMPHOMA
• Incidence higher in • Presence of Reed/Reed Sternberg Cells
males. o Diagnose through lymph node biopsy.
• Laboratory: o Sternberg cells in lymph node biopsy.
o Serum protein electrophoresis “M” spike in the gamma o Large, multinucleated cells with prominent large
globulin region. nucleoli (usually of B cell lineage).
o Marked rouleaux (increased). o Identifying characteristics of HL.
o Popcorn Cells: a variant of RS cells.

• Low – grade lymphoplasmacytic lymphoma.

• Excessive IgM (others decreased) (Paraproteins).
• Abnormal Ig interferes with platelet function and other
coagulation factors.
• Found in adults over 60 years old.
• Symptoms: nodal disease, hepatosplenomegaly, no BM
tumors.
• Laboratory:
o ESR
o Plasma Cells & Lymphocytes on blood smear
 MATURE LYMPHOID NEOPLASMS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

• Short term is NLPHL.

• B cell neoplasm.
• Characterized by “popcorn cells.”

• Sezary Cell
o Leukemic stage of MF.
➢ Sezary Syndrome: a variant of mycosis
fungiodes, presents as a disseminated disease
with widespread skin involvement & circulating
lymphoma cells.
o T cells with cerebriform Nucleus with a distinctive
folded, groovelike chromatin pattern.

• 40% of lymphomas.
• Seen in patient’s between 15 and 35 years old and over 55
years old.
• More frequent in males.
• Associated with EBV.
Subtypes (RYE Classification) BURKITT’S LYMPHOMA
• Nodular Sclerosis • Highly aggressive lymphoma of mature B cells.
o 70% are this subtype. • In the majority of patients, it is an extra-nodal lymphoma,
o Lowest EBV association. but some patients have leukemic manifestations.
• Mixed Cellularity • The 3 Epidemiological & Clinicopathological Variants:
o 20% are this subtype. endemic, sporadic, & HIV-related.
o Highest EBV Association • Endemic Burkitt’s Lymphoma occurs in equatorial Africa
• Lymphocyte Rich and in Papua New Guinea where malaria is hyperendemic.
• Lymphocyte Depleted • Strongly related to Epstein-Barr Virus (EBV) infection
❖ Take Note! All subtypes are associated with RSC. occurs in children and usually affects the jaw bones.
NON-HODGKIN’S LYMPHOMA • The lymphoma cells have the features described by the
• Involve mostly B lymphocytes. FAB group as L3/ALL.
• Malignant proliferation of lymphoid cells without Reed- • Correctly classified as a lymphoma (WHO
Sternberg cells. classification) since the cells are mature B cells.
• Neoplastic cells are medium sized with strongly basophilic
Classification Of NHL (WHO / REAL) vacuolated cytoplasm.
• Indolent (35 – 40% of NHL) • The vacuoles contain lipid and stain with Oil Red O.
o E.g., follicular lymphoma, small lymphocytic
lymphoma.
• Aggressive (-50% of NHL)
o E.g., diffuse large B-cell lymphoma.
• Highly Aggressive (-5% of NHL)
o E.g., Burkitt & Cutaneous T cell Lymphoma.

HAIRY CELL LEUKEMIA

• Most Common Type: Mycosis fungiodes • B Cell neoplasm.
• Associated with a lymphocytosis of Sezary cells. • Hairy Cells: later mature B cells expressing strong
• Mycosis Fungoides monotypic surface membrane immunoglobulins (IgM or
o It is caused by chronic antigenic stimulation IgG).
because of different exposures to: • Most of the patients are middle aged with a marked male
➢ Bacterial infections predominance.
➢ Smoking • Patients who present with advanced disease often shows
➢ Medications an immunodeficiency.
➢ Chronic sun exposure • Characterized by splenomegaly and cytopenia,
➢ Viral infections
➢ Chemical exposition
 MATURE LYMPHOID NEOPLASMS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

KEY DIFFERENTIATING FEATURES

Ovoid to indented to “kidney
bean” shaped; May be folded or
Nuclear Shape
dumbbell-shaped; usually central,
can be eccentric
Homogenous, fine to slightly
Chromatin
coarse; parachromatin is scant
One or more, small; rarely single
Nucleoli
and prominent
Moderate to abundant amount;
Cytoplasm
pale to gray-blue and agranular
The projections can be thick,
Fine (Hairy),
blunted, smudged, or serrated;
Irregular,
occasional cells may contain a
Filamentous
few, small vacuoles may be
Projections
present

HAIRY CELL
• Some cases may have lymphocytosis, rarely greater than
25 x 109/L.
• Positive staining with tartrate resistant acid
phosphatase (TRAP).
• The bone marrow aspiration often results in a “dry tap” due
to reticulin fibrosis.
• Splenomegaly is characteristic while lymphadenopathy
is usually minor.
• Absence of surface CD27 expression also distinguishes
hairy cell leukemia from other B-cell disorders.
• Clinical Features: splenomegaly and pancytopenia
CLINICAL FEATURES OF HAIRY CELL LEUKEMIA
• Painless superficial lymphadenopathy, usually >1 lymph
region.
• Usually presents as widespread disease.
• Constitutional symptoms (fever, weight loss, night sweats).
• Cytopenia: anemia ±neutropenia, ±thrombocytopenia if
bone marrow fails.
• Hepatosplenomegaly.
• Oropharyngeal involvement in 5 – 10% with sore throat and
obstructive apnea.
• Extranodal Involvement: most common
 TOPIC TITLE LOREM IPSUM
Ms. Aila Nell Sarmiento| 2nd Semester | A.Y. 2023-2024

OUTLINE
I. Secondary Hemostasis
II. Nomenclature of Procoagulants
III. Coagulation Groups
A. Based on Properties
i. Fibrinogen Group
ii. Prothrombin Group
iii. Contact Group
B. Based on Pathway
i. Extrinsic
ii. Intrinsic
iii. Common
IV. Coagulation Pathway
V. Thrombin Feedback

I. SECONDARY HEMOSTASIS
• Key player: COAGULATION FACTORS
o Other names: coagulation proteins,
procoagulants
o Produced mainly by the LIVER
✓ some factors are produced by
monocytes, endothelial cells,
megakaryocytes
• FORMS
o Enzymes (Zymogens -> Serine
protease/Transglutaminase)
✓ zymogen is inactivated enzyme;
when inactivated they become serine
proteases or transglutaminase
o (V and VIII) Cofactors: Enhances the
activity of enzymes
o Thrombin substrate: Factor I Fibrinogen
✓ a protein where thrombin binds to
o Plasma glycoproteins: Controls proteins that
avoid unnecessary blood clotting
✓ maintains balance in secondary
hemostasis
✓ ex of control protein: thrombomodulin
- prevents excessive production of
thrombin
II. NOMENCLATURE OF PROCOAGULANTS
• 1958, International Committee for the
Standardization of the Nomenclature of the Blood
Clotting Factors
• Named according to their order of initial
description/recovery
• Roman numerals
• “a” (activated) appears behind the numeral to denote
that the procoagulant has been activated
o Ex: Ia
o The zymogens include II, VII, IX, X, XI, XII,
and prekallikrein
o The serine proteases are IIa, VIIa, IXa, Xa,
XIa, XIIa, and kallikrein
• Factors I, II, III, IV - preferable called by their names
 TOPIC TITLE LOREM IPSUM
Ms. Aila Nell Sarmiento| 2nd Semester | A.Y. 2023-2024

• Adsorbed by BaSO4 and Al(OH)3

• VITAMIN K DEPENDENT
o Problems in Vit. K = clotting disorder
• CALCIUM DEPENDENT
• Reduced by oral anticoagulant
(Warfarin/Coumadin/Coumarin)
VITAMIN K
• A quinone found in green leafy vegetables
• Produced by intestinal bacteria:
o Bacteroides fragilis
o Escherichia coli
• Vit. K Dependent: *vit k is important in production of the ff*
o Procoagulants: II, VII, IX, X
o Regulatory Proteins: Protein C (destroys factor
V and VII), S, Z
• VIT. K IS DIFFERENT FROM POTASSIUM
• Possible causes of Vit. K deficiency:
1. Diet
2. Antibiotics
• For gamma carboxylation
o Enables the coagulation factors and
coagulation control proteins to bind ionic
calcium and cell membrane phospholipids,
especially phosphatidylserine
o Non-functional: Proteins induced by Vit. K
antagonists
• Causes of synthesis of non-functional factors:
o Vitamin K Deficiency
o Antibiotics
o Oral Anticoagulants
iii. Contact Group
• Activated via contact in foreign substances
• Members: XII, PK, HMWK, XI
• Produced in the liver
III. COAGULATION GROUPS
• Present in fresh plasma and serum
• Based on properties
• Activation: required contact with foreign surface
• Based on pathway
o in vivo
A. based on Properties
o in vitro
• Not based on roles on coagulation cascade
• VITAMIN K INDEPENDENT
• Important for LABORATORY ASSAYS
• CALCIUM DEPENDENT
1. Fibrinogen group
B. Based on Pathway
2. Vitamin K-Dependent Prothrombin group
• interdependent
3. Contact group
i. Fibrinogen Group • The pathways are not exclusive; all of them occur at
the same time
• Activated by thrombin
• They ALL work together to make fibrin clot
• Members: I,V, VIII, XIII
1. Intrinsic
• Completely consumed in clotting
2. Extrinsic
• Not present in serum – mixing studies (to identify 3. Common
missing coagulation factors i. Extrinsic Pathway
• Cleaved by thrombin • Tissue Factor Pathway
o fibrinogen converted by thrombin to fibrin clot
• Extrinsic “originating outside”
• Destroyed by plasmin
• initiated (started) by tissue factor, by which is
o plasmin: protein involved in fibrinolysis
normally outside the bloodstream or vascular system
• Found in platelets: I, V, VIII o tissue factor comes from the damaged blood
• VITAMIN K INDEPENDENT vessel lining that is found underneath the
• CALCIUM DEPENDENT vascular intima
• With highest molecular weight • Laboratory assay: Prothrombin Time
ii. Prothrombin Group ii. Intrinsic Pathway
• Members: II, VII, IX,X • Contact-Activation Pathway
• Produced in the liver • Intrinsic “originating within”
• Present in FRESH plasma and serum •
o Not present in adsorbed plasma •
 TOPIC TITLE LOREM IPSUM
Ms. Aila Nell Sarmiento| 2nd Semester | A.Y. 2023-2024

• involves coagulation factors circulating within the

bloodstream that are activated when they contact the
surface of certain cells
• Laboratory assay: Activated Partial Thromboplastin
Time (APTT)
iii. Common Pathway
● Links extrinsic and intrinsic pathways
● Final product: formation of fibrin clot

B. Intrinsic Pathway
• Factor XII activated by exposure to collagen
• Factor XIIa HMWK, & PK activate Factor IX
• IXa:VIIIa activates Factor X
• Factors involved: VIII, IX, XI, XII
• Complex formed: IXa:VIIIa (Intrinsic tenase)
o PK is activated by Factor XII to form Kall. Kall
will further activate Factor XII into Factor
XIIa. Factor XIIa will activate Factor XI to
Factor XIa. Factor XIa will activate Factor IX,
then Factor IXa will form a complex with
Factor VIII (IXa:VIIIa) which will also activate
Factor X.

IV. COAGULATION PATHWAY

A. Extrinsic Pathway
• Tissue Factor from injured blood vessel wall
activates factor VII
• TF: VIIa activates factor X
• Factors involved: TF (III), VII
• complex formed: TF:VIIa (Extrinsic tenase)
o TF activates Factor VII, they will form a
complex (TF:VIIa) that will activate Factor X.
After activation, Factor X will form a complex
with Factor V converting thrombin into
prothrombin– which is needed to convert
fibrinogen into fibrin polymer and eventually
the fibrin clot. Fibrin stabilizing factor is
Factor XIII.
 TOPIC TITLE LOREM IPSUM
Ms. Aila Nell Sarmiento| 2nd Semester | A.Y. 2023-2024

C. Common Pathway
• Both extrinsic and intrinsic pathway lead to the
activation of factor X
• Factors involved: X, V, II, I
• complex formed: Xa:Va (Prothrombinase)
• Xa:Va converts prothrombin (II) to thrombin (IIa)
• Thrombin cleaves fibrinogen (I) into fibrin & activates
factor XIII (fibrin stabilizing factor) to stabilize clot
Notes:
Thrombin is primary enzyme of secondary degree
hemodialysis
Plasmin is primary enzyme of fibrinolysis

V.THROMBIN FEEDBACK
• Mechanism or physiologic process that helps control
the degree of coagulation
• Low thrombin levels activate factors V, VIII (positive
feedback on the cascade), and XIII and induce
platelet aggregation
• When thrombin levels are high, thrombin binds to
thrombomodulin on the endothelial surface and
activates the protein C pathway
• Activated protein C and its cofactor, protein S,
inhibits factors V and VIII (negative feedback on the
cascade)

Effects of Bound Thrombomodulin to Thrombin

1. Loss of ability to activate factors V and VIII
2. Activates Protein C, leading to destruction of factors V
and VIII
3. Activates TAFI - fibrinolysis inhibitor
 MECHANISM OF SECONDARY HEMOSTASIS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

OUTLINE ❖ During coagulation, thrombin cleaves fibrinogen

generating a fibrin clot and activates factors V, VIII, XI,
I Coagulation Regulatory Mechanisms
and XIII propagating more thrombin generation.
A Tissue Factor Pathway Inhibitor (TFPI)
i 2 Steps of Coagulation Inhibition
• EC Protein C Receptor (ECPR)
B Protein C Regulatory System
o A transmembrane protein that binds protein C
i Steps of Protein C Regulatory System adjacent to the thrombomodulin-thrombin complex,
ii Protein C & S to activate PC (PC to APC).
iii Did You Know? Augments the action of thrombin-thrombomodulin at
C Antithrombin least fivefold in activating protein C to the serine
i Heparin protease APC.
ii Steps of Antithrombin • Protein S
D Other Serine Protease Inhibitors o Cofactor that binds and stabilizes Activated Protein C
(APC), is synthesized in the liver, and circulates in
plasma.
COAGULATION REGULATORY MECHANISMS
• Provide feedback loops to maintain a complex and delicate 2 Forms of Protein S
balance between abnormal thrombosis and bleeding.
Free Protein S
• Ensure localized coagulation (not systemic) and prevent Can serve as the APC cofactor
excessive clotting or thrombosis. (40%)
• Inhibitor deficiency leads to thrombosis or Bound Protein S -
-
thromboembolism that can block the blood vessels to C4bBP (60%)
cause death.
• Inhibitors (Natural Anticoagulant) Steps of Protein C Regulatory System
• Function to slow the activation of 1. Thrombin binds to thrombomodulin, activating the protein
Key Player C system.
procoagulants and suppress thrombin
production. 2. EPCR binds protein C adjacent to the thrombomodulin-
thrombin complex for activation.
• Tissue Factor Pathway Inhibitor (TFPI) 3. APC dissociates from EPCR and binds to Free Protein S.
Principal
• Antithrombin (AT) 4. APC-Protein S complex hydrolyzes and inactivates
Regulators factors Va and VIIIa, slowing or blocking thrombin
• Activated Protein C (APC)
generation and coagulation.
TISSUE FACTOR PATHWAY INHIBITOR (TFPI)
• Principal regulator of the Tissue Factor pathway or Extrinsic
pathway.
• It is synthesized primarily by endothelial cells (ECs) and is
also expressed on platelets.
• It is a Kunitz-type serine protease inhibitor and is the
principal regulator of the Tissue Factor or Extrinsic
pathway.
o Kunitz - 2: Binds to and inhibits Factor Xa.
o Kunitz - 1: Binds to and inhibits VIIa:TF complex
(Extrinsic Tenase)
• Enhanced by Protein S.
o Protein S: the cofactor of APC, is also a cofactor of
TFPI, and enhances factor Xa inhibition by TFPI
tenfold.

Steps of Coagulation Inhibition Protein C & S

1. TFPI first binds to Factor Xa and inactivates it. • Vitamin K - dependent regulator proteins.
a. Factor Xa is in the common pathway which is the • Protein C is activated when thrombin binds to
product of activation of the TF:VIIa complex. thrombomodulin on the endothelial cell surface.
2. TFPI:Xa complex binds and inactivates the cell bound • Both Inhibits Factor V and VIII to provide negative feedback
TF:VIIa complex, preventing more activation of Factor Xa. on the cascade.
Did You Know?
• Protein S-C4bBP binding is of particular interest in
inflammatory conditions because C4bBP is an acute
phase reactant.
• When the plasma C4bBP level increases, additional
protein S is bound and free protein S levels become
proportionally decreased, which may increase the risk of
PROTEIN C REGULATORY SYSTEM thrombosis.
• Activated by thrombin-thrombomodulin complex.
• Revises thrombin’s function from pro- to anticoagulant. Inflammation → ↑plasma C4bBP level → Binding of
o Thrombin: a primary enzyme of secondary protein S to C4bBP → ↓cofactor available & Protein C →
hemostasis, wherein in this method, it is used as a Thrombosis
facilitator to control its further production.
 MECHANISM OF SECONDARY HEMOSTASIS
Ms. Aila Nell C. Sarmiento, RMT, MSMT | 2nd Semester | A.Y. 2023-2024

ANTITHROMBIN • Potent inhibitor of Factor Xa,

• Produced in the liver. requires Protein Z, calcium,
• First identified coagulation regulatory protein, and first to ZPI (Z - dependent and phospholipid.
be assayed routinely. Protease Inhibitor) • Inhibits Factor XIa,
• It is the principal inhibitor of coagulation. enhanced by heparin.
• Inhibits the serine proteases (intrinsic pathway), including • Vitamin K dependent.
thrombin (factor IIa) and factors IXa, Xa, XIa, XIIa,
prekallikrein, and plasmin. • Found in plasma and other
• Enhanced by Heparin. body organs.
• Inhibits variety of proteases.
Heparin Protein C Inhibitor • Non-specific heparin-binding
• It enhances the action of antithrombin and is a member of serpin
glycosaminoglycan family of carbohydrates. • Protects Factor Va & VIIIa.
• Available from endothelium-associated mast cell granules
or as EC Heparan Sulfate. A1-protease
Inhibits Factor XIa, and it
❖ A cofactor or known as Heparin Cofactor II (HC II). inhibitor (Alpha 1
inactivates plasmin.
Antitrypsin)
Steps of Antithrombin
1. The heparin binds to antithrombin and changes its Alpha 2- Inhibits Thrombin, Factor Xa,
conformation or binding site. Macroglobulin Kallikrein, and Plasmin
2. AT binds to thrombin, forming Thrombin-Antithrombin- • Principal inhibitor of
Heparin complex (T-AT-Heparin). Alpha 2- Fibrinolysis.
3. T-AT detaches from heparin, is known as thrombin- Antiplasmin
• Inhibits free plasmin.
antithrombin (TAT) complexes which is the one measured
in the laboratory. • Important inhibitor of
Fibrinolysis.
Plasminogen • Secreted by endothelium.
Activator Inhibitor- • Inhibits plasminogen
1 (PAI-1) activator.
• Released from EC upon
damage.

OTHER SERINE PROTEASE INHIBITORS

 Laboratory Tests to Evaluate Secondary Hemostasis
Ms. Aila Nell Sarmiento | 2nd Semester | A.Y. 2023-2024

OUTLINE Arrow lets phlebotomist know whether the tube is filled in the
I. Other anticoagulants for Hemostasis minimum, middle, or maximum = All are acceptable due to
Specimen 10% error allowance
II. Needle Selection o Light blue top tubes carry 2.7 mL of blood with
III. Patient Management 0.3 mL anticoagulant = 3 mL
IV. Pre-Analytic Errors • Sodium Citrate volume adjustment for elevated
V. Specimen Collection Procedure hematocrits
A. Specimen Collection from Vascular Access o Polycythemia ‒ Hematocrit: 55% or higher ‒
Devices Raises anticoagulant-to-plasma ratio → Causes
B. Specimen Collection using Capillary o falsely prolonged results for clot-based
Puncture coagulation tests ‒ Amount of anticoagulant may
VI. Specimen Transport be computed by using this formula:
VII. Specimen Handling and Processing
A. Sample Testing and Storage
B. Centrifugation
C. Platelet-Poor Plasma C = Volume of 3.2% Sodium Citrate in mL
VIII. Platelet-Poor Plasma For Clot-Based Testing V = (Desired) Volume of Whole Blood + Sodium Citrate in mL
IX. Specimen Storage H = Hematocrit percent
X. Specimen Rejection I. OTHER ANTICOAGULANTS FOR HEMOSTASIS
SPECIMEN
LABORATORY INVESTIGATION OF SECONDARY 1. Ethylenediaminetetraacetic Acid (EDTA)
HEMOSTASIS ▪ For CBC including platelet count
Evaluate: ▪ Factor V Leiden mutation
1. Coagulation Factors 2. Molecular Testing
2. Inhibitors of Coagulation ▪ Acid citrate dextrose (ACD, yellow closure)
• Hemostasis Specimen Collection Tubes ▪ K2EDTA (white closure)
o Plastic or siliconized light blue-closure tubes 3. Heparin
✓ Plastic is a non-contact surface. ▪ Never been validated for plasma coagulation
✓ Glass tubes are available but its use is testing
declining due to potential breakage ▪ May be used for platelet counts in cases of
concern with risk of exposure to platelet satellitosis (satellitism)
bloodborne pathogens. 4. Citrate Theophylline Adenosine Dipyridamole (CTAD)
o Buffered 0.105-0.109 M (3.2%) Sodium Citrate ▪ Blue Closure
✓ Mechanism: chelates Calcium ▪ Suppress in vitro platelet or coagulation
✓ “Buffered” and Maintaining cap of tube: activation for specialty assays
Maintain pH of specimen ✓ Platelet Factor 4 (PF4): platelet
o Recommended by NCCLS activation marker
o Ratio of Anticoagulant: Blood = 1:9 +/- 10% ✓ Thrombin-Antithrombin Complex (TAT):
✓ The ratio is important because once you coagulation activation marker
mess up with the ratio, it could affect ▪ Ideal for patient undergoing anticoagulant
the mixing of the sample with the therapy
reagents in coagulation testing. II. NEEDLE SELECTION
o Adjust blood/citrate volume when patient
hematocrit is >55%

• Choose right needle to avoid hemolysis

 Laboratory Tests to Evaluate Secondary Hemostasis
Ms. Aila Nell Sarmiento | 2nd Semester | A.Y. 2023-2024

III. PATIENT MANAGEMENT 9. Specimen Storage >25 C

• No fasting requirements • Factor V and VIII deteriorates
• Avoid caffeine and exercise for 2 hours
• No smoking 30 minutes before collection
• Inactive patient for 5 minutes before blood collection

IV. PRE-ANALYTIC ERRORS

1. Partial Fill Tubes (Short Draw)
• Falsely prolonged clot-based coagulation test results
• Specimen with a smaller volume than the minimum
specified by the manufacturer V. SPECIMEN COLLECTION PROCEDURE
• Excess anticoagulant relative to blood volume • Follow the order of draw
adversely neutralizes test reagent Calcium (CaCl2) ✓ If a series of evacuated tubes is to be filled
• Remedy: Discard and Recollect from a single venipuncture site, hemostasis
✓ Most plastic blue-topped tubes collect 2.7 specimens must be collected first or
mL of whole blood (0.3 mL anticoagulant) immediately after a nonadditive tube.
✓ Blood collected must be within 90% of the ❖ If hemostasis tube after additive tube, additives may
calibrated volume transfer to hemostasis specimen and invalidate test
✓ Smaller tube = narrower tolerance for short results.
draws ✓ If non-additive tubes are unavailable, use
2. Vacuum Leak & Citrate evaporation and discard a preliminary blue-closure tube
• When tube falls on floor when following additive tubes.
• When trying to open rubber stopper of tube ✓ Order of draw for evacuated and syringe
• Citrate Evaporation - when using expired evacuated system:
tube 1. Yellow tube
3. Specimen Clot 2. Light blue tube
• Falsely shortening 3. Serum tube
4. Visible Hemolysis 4. Green tube
• Falsely shortening 5. Lavender tube
• Interferes with optical endpoint coagulometer results 6. Gray tube
• Cocktail shaking: Excessive specimen agitation causes A. Specimen Collection from Vascular Access Devices
hemolysis (RBC rupture) • Vascular access device blood collection
5. Lipemia / Icterus ✓ Performed by physicians and nurses
• Interferes with chromogenic substrate methods ✓ Before blood is collected for hemostasis
• Remedy: Use electro-mechanical detection method testing, the line must be flushed with 5 mL of
instrument Saline
6. Prolonged Tourniquet Application Do not flush with Heparin
• Venous Stasis: Falsely shortening ✓ The first 5 mL of blood, or 6x the volume of blood
✓ Results from local accumulation of vWF and collection tube, must be collected and discarded
Factor VIII ✓ Blood is collected into a syringe and transferred to an
• Activates EC evacuated tube using a safety transfer device
• Elevates concentration of vWF and Fibrinogen
• Hemoconcentration
7. Traumatic Venipuncture
• Falsely Shortening
• Due to “fishing”: Cannot find vein of patient
• Activation of Extrinsic Pathway
8. Specimen Storage at 1-6 C
• Precipitation of vWF
• Activation of Factor VII and XI
• Activation of platelets
• Destruction of platelet integrity
 Laboratory Tests to Evaluate Secondary Hemostasis
Ms. Aila Nell Sarmiento | 2nd Semester | A.Y. 2023-2024

B. Specimen Collection Using Capillary Puncture ✓ Release of PF4 leads to falsely shortening
• Follow procedure for capillary puncture the PTT and interfere with Heparin
• Notes: management
✓ The blood collector places collection device IX. SPECIMEN STORAGE
adjacent to the free-flowing blood and • Storage Temperature: 15-25 degrees Celsius
allows the device to fill by capillary action
and gravity
✓ The key to accurate capillary PT/INR
measurement is a free-flowing puncture
VI. SPECIMEN TRANSPORT • If coagulation test cannot be completed within
• Specimens are placed in a rack and allowed to stand prescribed interval, immediately centrifugate and
in a vertical position with the closure intact and quick freeze
uppermost • Thaw frozen sample at 37 C before testing
• Maintaining the blood collection tube seal minimizes • Do not re-freeze sample
CO2 diffusion X. SPECIMEN REJECTION
✓ Otherwise, causes pH to rise, falsely • The following are reasons for rejecting collected
prolonged PT and PTT specimens:
• Visual checks 1. Specimens lacking proper identification
✓ Fibrin clot 2. Hemolyzed blood sample
✓ Hemolysis 3. Incorrect tube for specimen
✓ Correct sample volume 4. Improper handling of specimen
VII. SPECIMEN HANDLING AND PROCESSING 5. Contamination of specimen
A. Sample Testing and Storage 6. Inadequate sample to perform test, referred
• PT = 24 hours to as “Quantity not Sufficient”(QNS)
7. Incorrect time of specimen collection
• APTT = 4 hours
8. Clotted specimens in an anticoagulated tube
✓ APTT for Unfractionated Heparin (UFH)
9. No date or time marked on label
Therapy: centrifuge within 1 hour
10. Label information does not match the
• Uncentrifuged/centrifuged
requisition
• 15-25 degrees Celsius
B. Centrifugation
• Within 1 hour of collection
• Minimum 1500x g for 15 minutes
• Separate plasma from red cells with plastic pipette,
then to a plastic tube/ cup
C. Platelet-Poor Plasma
• <10,000 uL
VIII. PLATELET-POOR PLASMA FOR CLOT-BASED TESTING
• Platelet Poor Plasma (PPP)
• Sample for clot-based coagulation testing
• Platelet count: Less than 10, 000/ uL
• How to prepare:
✓ Centrifugation: 1500x g RCF for 15 minutes in
a horizontal-head centrifuge
• Effects of PPP with >10,000/ uL platelets
✓ Platelets become activated in vitro and
release microparticles and the membrane
phospholipid Phosphatidylserine, which
triggers plasma coagulation and interfere
with Lupus Anticoagulant (LAC) testing
✓ Desensitization of PT and PTT and interfere
with clot-based coagulation assays
Laboratory Tests to Evaluate Secondary Hemostasis
Ms. Aila Nell Sarmiento | 2nd Semester | A.Y. 2023-2024
 LESSON 8: Tests to Evaluate Secondary
nd
Ms. Aila Nell Sarmiento | 2 Semester | A.Y. 2023-2024
Hemostasis
Procedure:
OUTLINE 1. Label 3 tubes (1, 2, 3) and place in a water bath (37 C)
2. Place 1 mL of blood into the three tubes
3. Start timer as soon as 1 mL of blood enters tube 1
4. Gently tilt tube 3 every 30 seconds and observe for
presence of clot
5. After clotting is observed in tube 3, observe tube 2.
When clotting is observed in tube 2, observe tube 1.
6. Stop timer when no flowing of blood is observed in
tube 1.

OVERVIEW ON CLOTTING TIME

• performed as a partner for bleeding time
• bleeding time: evaluate bleeding function
✓ clotting time is more on evaluating secondary
hemostasis
• Two methods: • Reference Range: 7-15 minutes
✓ Slide method (drop method)
✓ Tube method (lee-white whole blood clotting II. CLOTTING TIME
time) Slide or Drop Method
• Sample obtained from capillary puncture
LEE-WHITE WHOLE-BLOOD COAGULATION TIME TEST Procedure:
• First laboratory procedure designed to assess 1. Perform skin puncture
coagulation 2. Wipe 1st drop of blood. Will cause falsely prolonged
• First in vitro clot procedure with principle that the time clotting time if not wiped.
interval from the initiation of clotting to visible clot 3. Place 2nd drop of blood to the center of glass slide
formation reflects the condition of the coagulation 4. Pass a tip of needle to the drop of blood every 30
mechanism seconds
• Principle: To measure how long it takes for blood to 5. Stop timer as soon as fibrin strands were seen
clot • Reference Range: 2-4 minutes
✓ place a sample in a test tube and perform the III. ACTIVATED CLOTTING TIME
test at 37 C using water bath • Developed by Dr. Paul Hattersley
✓ the principle is simple but it become a basis • Widely used point-of-care assay to monitor:
for many coagulation tests 1. UFH (heparin) therapy
• Prolonged CT = Coagulopathy/coagulation deficiency 2. Cardiac catheterization
3. Coronary artery bypass graft surgery
Basis of standard clot-based coagulation screening tests • Activator: Diatomaceous Earth (Diatomite)
1. Prothrombin time • Equipment: Tubes with diatomite
2. Partial thromboplastin time • Maintained at 37 C
3. Fibrinogen assay
4. Thrombin clotting time
5. Coagulation factor assays
6. Tests of Fibrinolysis
7. Inhibitor assays
8. Reptilase time
9. Russel Viper Venom Time
 LESSON 8: Tests to Evaluate Secondary
nd
Ms. Aila Nell Sarmiento | 2 Semester | A.Y. 2023-2024
Hemostasis
• Reference Range: 75-120 seconds MNEMONIC
✓ Heparin Therapy: 140-185 seconds • Extrinsic: TF (III), VII
Significance: ✓ 3 + 7 = 10
• Prolonged ACT = Indicative of one or more factor • Intrinsic: XII, XI, IX, VIII
defects in the Intrinsic or Common Pathway or ✓ Twelve, Eleven, Nine, Eight (TENe = 10)
presence of circulating anticoagulant such as Heparin • Common: X,V, Prothrombin (II), Fibrinogen (I)
✓ 5 x 2 x 1 = 10 (factors of 10)
EVALUATION OF THE COAGULATION • Vit. K Dependent: II, VII, IX, X
• End point of most coagulation tests: Formation of ✓ 2 + 7 = 9, after 9 is 10
fibrin clot
Prothrombin Time (PT) A. Prothrombin Time
• Extrinsic & Common Pathway Reagent: Tissue Thromboplastin
✓ Play Tennis → OUTSIDE o consists of recombinant or affinity-purified=
Partial Thromboplastin Time (PTT) tissue factor suspended in phospholipids mixed
• Intrinsic & Common Pathway with a buffered 0.025 M solution of calcium
✓ Play Table Tennis → INSIDE chloride
Thrombin Time • Sodium citrate reversibly chelates calcium (reversibly
• Fibrinogen prevents coagulation) so upon the addition of fresh
supply of calcium ions, it can override the
QUICK RECAP anticoagulant → coagulation will take place
Extrinsic Pathway o Few thromboplastins are organic extracts of
• activated by exposure of tissue factor– found in emulsified rabbit brain or lung suspended in
smooth muscles and fibroblast; an extravascular calcium chloride
component that activates the coagulation cascade • Extrinsic & Common Pathway
Intrinsic Pathway • Factors involved: VII, I, II, V, X
• All of procoagulants are found inside the circulation
• activated by surface contact
Common Pathway
• end-product of both extrinsic and intrinsic pathway
activates factor X In PT and APTT, these events are
being replicated (in vitro) to further have a close-up
look at the factors involved
 LESSON 8: Tests to Evaluate Secondary
nd
Ms. Aila Nell Sarmiento | 2 Semester | A.Y. 2023-2024
Hemostasis
i. Procedure: PT ✓ The most sensitive thromboplastin reagents have an ISI
1. The tissue factor-phospholipid-calcium chloride reagent is value of 1.00, based on World Health Organizations
warmed to 37 C (WHO) standards
2. An aliquot of test PPP (Platelet Poor Plasma), 50 to 100 uL is
transferred to the reaction vessel iii. Causes of Prolonged PT
✓ Sample: PPP During oral anticoagulant therapy
3. The PPP is incubated at 37 C for 3 to 10 mins. • Congenital factor deficiencies (factor X, VII, V;
4. 100 to 200 uL of reagent is directly added profound prothrombin deficiency; and fibrinogen)
5. As the clot forms, the timer stops, and the elapsed time is ✓ I, II, V, X, VII
recorded • Circulating anticoagulant
• Multiple factor deficiencies (DIC, liver disease, Vit. K
NOTE: deficiencies
- After the addition of CaCl2 reagent: begin ✓ Vit. K is important in producing functional
timing for clot formation coagulation factors such as II, VII, IX, X (for PT:
- Test must be performed at 37 degree celsius II, VII, X)
- Equipment used: Coagulometer ✓ liver is responsible for production of
ii. Reporting of PT Results coagulation factors
Report to the nearest tenth of a second ✓ Disseminated Intravascular Coagulation (DIC)
• Reference Range: 12.6 to 14.6 secs. wherein the supply of coagulation factors are
✓ established by each institutions depleted
• INR value - International Normalized Ratio
✓ Used for Coumadin/Warfarin monitoring To distinguish between Vit. K deficiency from liver disease, the
✓ Reported together with PT following tests may be performed:
✓ Means of standardizing the PT and eliminates 1. Factor V assay
the difference seen in laboratories 2. Factor VII assay
thromboplastin reagent ➢ both factor V and VII are low → liver disease
INR – International Normalized ratio ➢ factor V is normal and factor VII is low → Vit. K
• No reference range deficiency
✓ The therapeutic range is dependent on the B. Activated Partial Thromboplastin Time
condition being treated, but it is generally • Addition of Surface activator + exogenous
considered to be between 2.0 to 3.0. phospholipid + Ca++ ion supply
✓ Can also be used to adjust the dosage of Main Reagent:
coumadin/warfarin 1. Phospholipid (Prev. called partial thromboplastin or
✓ High INR → slow clotting cephalin)
✓ Low INR → fast clotting 2. Negatively charged particulate activator
a. Silica
b. Ellagic Acid
c. Celite
d. Kaolin
3. Calcium chloride
• Intrinsic & Common Pathway
• Factors involved: I, II, V, X, VIII, IX, XI, XII (PK, HK)
i. Procedure: APTT
1. 50 to 100 uL prewarmed (37C) reagent (PLT + activator) is
mixed with equal volume of PPP.
2. Incubate for exactly 3 minutes.
3. Add 50 to 100 uL prewarmed 0.025 M CaCl then start the
timer.
ISI - International Sensitivity Index 4. Stop timer once fibrin is produced and record the time
✓ Related to the thromboplastin reagent interval.
✓ This number is provided by the manufacturer and is lot Reference range: 26-38 seconds
number and instrument specific
 LESSON 8: Tests to Evaluate Secondary
nd
Ms. Aila Nell Sarmiento | 2 Semester | A.Y. 2023-2024
Hemostasis
NOTE: D. Thrombin Time (Thrombin Clotting Time)
- also uses Coagulometer • Addition of exogenous thrombin + Ca++ ion supply
- reference range can also vary depending on the reagent and • Focuses only in conversion of fibrinogen to fibrin clot
equipment that is used • Very good test to detect deficiency in fibrinogen
• Reagent: Thrombin at 5 NIH units/mL
ii. Clinical Application of APTT • Thrombin cleaves fibrinopeptides A and B to form
• Standard method for monitoring unfractionated fibrin polymer
heparin • Prolonged TCT:
• Detects the presence of LAC (Lupus Anticoagulant) ✓ Afibrinogenemia
• Detects deficiencies of one or more coagulation ✓ Hypofibrinogenemia (<100 mg/dL)
factors: prothrombin(II); factor V, VIII, IX, X, XI, or XII; ✓ Dysfibrinogenemia
or fibrinogen (I) when fibrinogen level is 100 mg/dL or ✓ Presence of antithrombotic material (FDP,
less paraproteins, heparin)
• Deficiencies in factor XII, PK, and HMWK cause • Dabigatran: oral direct thrombin inhibitor
prolonged APTT but DOES NOT cause bleeding ✓ quantitative measurement may be performed
• Prolonged in the presence of anti-factor VIII, anti- using plasma-diluted TCT
factor IX, LAC, FDP, and paraproteins.
• Prolonged in Vit. K deficiency, DIC and Rosenthal i. Procedure: TCT
syndrome 1. Thrombin reagent is warmed at 37C for 3-10 minutes.
C. Differential Diagnosis of PT and PTT 2. An aliquot, usually 100 uL of normal PPP isincubated for 3-10
• PT and PTT are normal mins.
✓ Platelet deficiency, vascular defect, f XIII 3. Add 200 uL of thrombin into PPP aliquot and start timer
abnormality 4. Timer is stopped once clot is formed
✓ Coagulation factor is not the problem Reference range: 15-20 seconds
✓ PT and PTT are not sensitive to f XIII (use a
different assay for it) REPTILASE TIME
• PT is prolonged and APTT is normal • thrombin-like enzyme isolated from the venom of
✓ Factor VII deficiency Bothrops atrox snake
✓ Factors in common pathway is not deficient IF • Cleaves only fibrinopeptide A
APTT is NORMAL • Same as TCT but uses Atroxin as reagent
• PT is normal and APTT is prolonged ✓ detects deficiency in fibrinogen
✓ problem within the intrinsic pathway • unaffected by heparin
✓ associated with bleeding: VIII, IX, and XI ✓ TCT is affected by UFH (unfractionated
defects heparin therapy), so if you want to determine
✓ Not associated with bleeding: XII, PK, HMWK, the patient’s fibrinogen adequacy and they
LAC are undergoing heparin therapy, TCT cannot
• PT and PTT are prolonged be used because heparin can interfere with
✓ abnormality in common pathway the result.
✓ Medical conditions: anticoagulant, • Atroxin is reconstituted with d. H2O and is stable for
disseminated intravascular coagulation (DIC), 1 month when stored 1-6 C
liver disease, Wit. K deficiency, massive ✓ Note: Poisonous and is fatal if enters the
transfusion bloodstream
✓ rarely dysfibrinogenemia • Reference range: 10-15 seconds
✓ factor X, V, II defects

❖ LET’S PRACTICE!
Determine the possible PT and APTT result for the following
deficiencies:
1. F XII def - Prolonged APTT; Normal PT
2. F VIII def - Prolonged APTT; Normal PT
3. F VII def - Prolonged PT; Normal APTT
4. F XI def - Prolonged APTT; Normal PT
5. F X def - Prolonged APTT & PT
 LESSON 8: Tests to Evaluate Secondary
nd
Ms. Aila Nell Sarmiento | 2 Semester | A.Y. 2023-2024
Hemostasis
III. RUSSEL VIPER VENOM TIME B. Single Factor Assay
• AKA Stypven Time (obsolete) • Used to confirm a suspected factor deficiency, as
• Venom from: Daboia russelii viper suggested by mixing study that shows correction
• Triggers coagulation at Factor X • Used in:
• Detects the coagulation factor deficiency involved in ✓ Factor VIII def. (Hemophilia A)
common pathway ✓ Factor IX def. (Hemophilia B)
• Differentiates FVII and FX deficiencies ✓ Factor XI def. (Rosenthal syndrome)
✓ PT detects both extrinsic and common factor • Clinical use: Monitoring/documentation therapy
deficiency and Stypven time only detects • Reporting or result: percentage of factor activity
common pathway factors
✓ PT: prolonged; Sytpven time: normal → VI. CIRCULATING INHIBITORS/ CIRCULATING
ANTICOAGULANTS
EXTRINSIC PATHWAY (FVII) Detected by the prolongation of PT or APTT which is not
✓ PT: prolonged; Stypven time: prolonged → corrected by addition of fresh plasma
COMMON PATHWAY • Non-specific inhibitors
• Buffer-diluted russel viper venom time (dRVVT) ✓ Targets Phospholipid-Protein Complex
✓ modified RVVT ✓ ex: Lupus anticoagulant: IgG immunoglobulin
✓ used to detect lupus anticoagulant directed against a number of phospholipid-
protein complexes (Xa-Va-Calcium-plt
IV. DUCKER’S TEST/5M UREA SOLUBILITY TEST phospholipid)
• test specific for F XIII (fibrin stabilizing factor) • Specific inhibitors: IgG immunoglobulins directed
deficiency against coagulation factors
• F XIII function - gives stable fibrin clot ✓ ex: Anti-factor VIII
• Patient sample + CaCl2 → induce coagulation ▪ the most common of the specific
• if F XIII is present, the clot that will be formed is stable, inhibitors
and even with the addition of 5M urea, the clot will ▪ detected in 10-20% of patients with
remain stable. severe hemophilia
• if F XIII is absent/deficient, the clot that will be formed ▪ Hemophilia A
is NOT stable, and with the addition 5M urea, the clot ✓ ex: Anti-factor IX
will be dissolved immediately/disintegrated ▪ detected in 1-3% of factor IX-
COAGULATION FACTOR ASSAYS deficient patients
Assays that target specifically select coagulation factors ▪ Hemophilia B
A. Fibrinogen
• Fibrinogen (Factor I)
Elevated in:
✓ Pregnancy
✓ Inflammatory states
✓ Stress
✓ Oral contraceptives
✓ Liver disease (moderately severe)
- Hypofibrinogenemia (<200 mg/dL)
- seen in DIC and severe liver disease

Methods of Measurement:
• Precipitation and denaturation
• Turbidimetric or fibrin clot density PTT MIXING STUDIES (for inhibitors)
• Coagulable protein assay 1. LAC
2. Specific Factors Inhibitors
• Immunologic assays
3. Factor deficiency
• False decrease fibrinogen result
✓ seen in patients that has Heparin level >0.6
U/mL
✓ FDP levels>100 microgram/mL
Results are reported as mg/dL of fibrinogen
 LESSON 8: Tests to Evaluate Secondary
nd
Ms. Aila Nell Sarmiento | 2 Semester | A.Y. 2023-2024
Hemostasis
Detecting LAC and Specific Inhibitors
1. TCT is performed to rule out heparin
✓ TCT: prolonged = heparin
2. If no heparin is present, the patient's PPP is mixed
with an equal amount of pooled normal PPP, and PTT
is performed.
Note: If the mixture PTT corrects to within 10% of the PNP PTT
(or to within the reference interval) and the patient is
experiencing bleeding, a coagulation factor deficiency
(coagulopathy) is presumed.

3. 2nd aliquot of pt. PPP and rgt. PPP is incubated for 1-2
hrs. at 37 C
Note: If after 1-2 hrs. incubation, the PTT result is corrected, the
originally prolonged PTT result is caused by factor deficiency

NIJMEGEN-BETHESDA TITER FOR ANTI-FACTOR VIII INHIBITOR

• Used to confirm and quantify specific anti-factor VIII
inhibitor
• Quantitated by mixing the test plasma with known
Factor VIII
• After incubation, residual Factor VIII is measured by
specific factor assay
 LESSON 8: Tests to Evaluate Secondary
nd
Ms. Aila Nell Sarmiento | 2 Semester | A.Y. 2023-2024
Hemostasis
• Principle: Check for the presence of an inhibitor by
allowing it first to consume the known Factor VIII and
then checking for the residual factor VIII.
✓ If there’s consumption of F VIII without
addition of any reagent → presence of
inhibitor
✓ if there’s no consumption after incubation →
no inhibitor present
• Amount of inhibitor present: calculated by comparing
the VIII activity in the Px incubation mixture with that
of the control mixture
• Additional note:
✓ Lower value of residual factor VIII indicates
presence of inhibitor
✓ No changes means inhibitor is absent