Basic Concepts of Hematology (Hemostasis)

BASIC CONCEPTS OF HEMATOLOGY
Hemostasis
Objectives are indicated by number. The student upon completion of the
classroom component of the hemostasis section will be responsible to
successfully:
01
EXPLAIN THE BASIC CONCEPTS / PURPOSES OF HEMOSTASIS.
Hemostasis (hemo = blood + stasis = stop, cease) is a process by which the body
maintains the life flow of blood and prevents bleeding problems. Hemostasis is
a complex sequence of interactions in the (1) blood vessels, (2) thrombocytes,
(3) coagulation proteins of blood, and (4) fibrinolytic proteins of blood. This
mechanism is able to retain blood within the injured vessel until the repair is
completed. The end product of hemostasis is the transformation of blood into a
thrombus or clot. This is the culmination of a three step phenomenon
consisting of (1) extravascular, (2) vascular, and (3) intravascular phases.
02 LIST FIVE FACTORS WHICH AFFECT THE EFFECTIVENESS OR
INEFFECTIVENESS OF HEMOSTASIS.
[1] Type of injury, bruise, cut, abrasion, etc.
[2] Magnitude of the injury.
[3] Size of the vessels involved and their ability to respond.
[4] Hydrostatic pressures within the blood vessels and tissues.
[5] Availability, quantity, and quality of platelets, clotting factors, and
inhibitors.
03
DESCRIBE AND/OR ILLUSTRATE THE THROMBOCYTE.
The thrombocyte (platelet) is an active sol-gel structure capable of

undergoing rapid morphological changes. The platelet consists of four distinct
zones (1) peripheral zone, (2) sol-gel zone, (3) organelle zone, and [4]
membrane zone. Each zone has a unique function to be covered in the
following objectives. Review the following illustration which displays a cross
section of a platelet.
04 DISCUSS THE PERIPHERAL ZONE AND ITS FUNCTIONAL ROLE IN THE

PLATELET.
The peripheral zone consists of the exterior coat or glycocalyx, platelet
membrane, surface connecting channels (open canalicular system), and
submembranous region. The glycocalyx contains a variety of glycoproteins that
constitute the ABO antigens and human leukocyte antigens (HLA). The
glycoproteins function as receptors and stimuli transmitters across the platelet
membrane. The platelet membrane is also made up of lipoproteins,
phospholipids, and glycolipids. These molecules function as receptors for (1)
fibrinogen, (2) fibronectin, (3) vonWillebrand Factor, (4) epinephrine (5)
thrombin, (6) ADP, and (7) serotonin. Platelet factors PF1 - PF7 along with
coagulation factors V and VIII are found on the platelet membrane. The open
canicular system serves as a means for releasing platelet stored products to the
outside.
A. The Glycocalyx is the fluffy coat type of cell membrane, consisting of
plasma proteins, carbohydrates, and other molecules. The molecules are
related to coagulation, complement, and fibrinolytic activities. The glycocalyx
also extend into the interior channels.
B. The submembranous region, along with the open canalicular system forms a
sponge-like area t`hat facilitates selective absorption of clotting factors.
C. This membrane zone contains the following distinctive molecules:
a. Arachidonic acid, a major component of the membranes
phospholipid structure.
b. Glycoprotein Ib, a receptor molecule for vonWillebrands factor.

c. Glycoprotein IIa/IIIa complex, a receptor for fibrinogen.
d. Phospholipids (phosphatidylcholine, phosphatidylethanolamine,
phosphatidylinositol, and sphingomyelin)
05
DISCUSS THE GEL-SOL ZONE AND ITS FUNCTION IN THE PLATELET.
This mid-zone structure forms the cytoskeleton of the platelet. It may be

referred to as the structural zone and contains a matrix of microtubules,
microfilaments, and submembranous filaments. The submembranous filaments
extend into the peripheral zone.
A. The microtubules are found throughout the platelet and help to conform to
it discoid shape. The microtubules have contractile capability and can cause
movement of the organelles to facilitate platelet secretion activity. The
contractile ability of the microtubule enable the tubules to concentrate in
various regions of the platelet. These structures regulate secretion responses of
the platelet in addition to its skeletal role.
B. The microfilaments are composed of thrombosthenin, actin, and myosin
filaments. These contractile proteins make up the major portion of the platelet
proteins. They are found throughout the interior of the platelet. They exist in
an unorganized gelatinous state and upon proper stimulus they will organize
into a definite structure to cause platelet contraction.
C. Submembranous filaments are specialized microfilaments that link the outer
platelet membrane to the microfilaments in the sol-gel area. These may
facilitate organizing the platelets shape and to affect the granules.
The primary function of the sol-gel zone is to provide a cytoskeleton for the
platelet and enable it to contract to release platelet secretions and enable the
formation of the pseudopods involved in the coagulation process.
06
DISCUSS THE ORGANELLE ZONE AND EXPLAIN ITS FUNCTION.
The metabolic activities of the platelets are carried out in this innermost zone.
The major structures in the organelle zone are (1) dense granules, (2) alpha
granules, (3) peroxisomes, (4) mitochondria, (5) glycogen, and (6) lysosomes.
A. Alpha granules in a single platelet range form 20 to 200 in number. These
granules contain a wide assortment of molecules: (1) platelet factors 1-7, (2)
-thromboglobulin, (3) basic protein, (4) platelet-derived growth factor, (5)
fibrinogen, (6) vWF, (7) factor V, (8) albumin, (9) fibronectin, (10)
plasminogen, (11) high-molecular-weight kininogen, (12) protein S, (13) IgG,
(14) osetonectin, and (15) thrombospondin. The platelet can release these
chemicals through vascular injury.
B. Dense granules, so called because of their opaque behavior under the
electron microscope, range in number from two to twelve in a single platelet.
Dense granules are known to contain (1) ADP, (2) ATP, (3) calcium, (4)
epinephrine, (5) norepinephrine, (6) serotonin, (7) pyro-phosphate, and (8)
magnesium. The platelet can release these chemicals during vascular injury.
C. Lysosomes contain acid hydrolases.

D. Mitochondria are energy production organelles, numbering from 10 to 64
per platelet. Platelets require energy to function in hemostasis. Platelet ATP is
generated by glycolysis and the Krebs cycle.
07 DESCRIBE THE MEMBRANE SYSTEM OF THE PLATELET.
This is a structural zone that consists of a dense tubular system and an open
canicular system (OCS) that connect the outer surface of the platelet to its
inner regions. These two tubular systems can fuse to form a membrane
complex. The principle role is associated with calcium ions (storage and
release).
08
DESCRIBE HOW PLATELET ACTIVATION IS INITIATED.
Activation can be either transient, reversible, or irreversible. Platelet

activation causes the platelet to respond in a graded fashion that includes
stickiness, adhesion, shape changes, release of platelet substances, and
aggregation. The strength and duration of the stimulus determines the platelet
response. Another factor that affects the platelets activation is the
physiological health of the platelet. When the platelet is activated, pseudopods
will form, and the platelet begins to contract. Factors that will cause platelet
activation are (1) exposure to collagen from vascular injury, (2) ADP, (3)
platelet activating factor, (4) thrombin, (5) epinephrine, (6) thromboxane A.
NOTE
[1] Thromboxane A is a prostaglandin that can cause the formation of
thrombi. It will cause platelet aggregation.
[2] Platelet activation factor is produced and released by monocytes
and macrophages that can cause platelets to aggregate. Its chemical
name is 1-alkyl-2-acetylglycerophospho-choline.
[3] Thrombin is activated prothrombin and is part of the coagulation
scheme.
[4] Epinephrine is a hormone known as adrenalin and is the principle
that initiates the flight-fight mechanism. In addition to platelet
aggregation, it can cause fat breakdown, increase strength of the
heartbeat, inhibits glycogen synthesis, and stimulate glycogenolysis.
[5] ADP is adenosine diphosphate, a molecule that can accept a
phosphate group to form adenosine triphosphate (ATP) and serve as
the energy currency of the cell. It causes the platelet membrane to
:shift and exposes receptors.
[6] Collagen is a major water insoluble protein of the white fibers of

connective tissue.
Platelet Fact
Platelets remain disc-shaped and inert in a normal vascular environment. When
the vascular epithelium is disrupted/altered, exposing collagen, platelets
become activated and the formation of the platelet plug begins. The following
sequence of events occur: (1) adhesion, (2) activation, (3) primary aggregation,
(4) secretion, and (5) secondary aggregation. This is a cycling event and results
in the formation of a mechanical barrier over the injury.
09
DISCUSS PLATELET ADHESION.
Adhesion occurs when a collagen fiber is exposed. Platelets attach to the

foreign surface of the collagen. The attachment of the platelet required the
ability of the vonWillebrands factor (vWF) to bind to collagen or appropriate
damaged surface and the binding of vWF molecules to the glycoprotein Ib
molecules of the platelet membrane. Platelets will layer to the injury by this
mechanism. The contact of the platelet membrane causes morphological and
functional changes in the platelet known as activation.
10
DISCUSS PLATELET ACTIVATION.
There are three things that characterize platelet activation: (1) change in
shape, (2) development of surface receptors, and (3) changes in phospholipid
orientation in the membrane. A transformation occurs in which the dis-shaped
platelet becomes spherical and tiny projections begin to extend from the
membrane surface (pseudopods or microfilaments). These projections increases
the interaction between platelets. The formation of the pseudopods causes the
secretory granules and other organelles to draw closer to the membrane
surface. More platelets will adhere to each other and any exposed collagen, to
form a covering film of protection. This has been described in textbooks as a
jigsaw puzzle effect.
The process requires the presence of surface glycoproteins which forms the
actual bonds with the exposed epithelium. One of the glycoproteins, von
Willebrand factor acts as a bridging molecule between the platelet molecule
and the site of injury. The adhesion phenomenon begins within 1 - 2 minutes of
injury. If the activation process is acute enough, secretory activity will begin.
The other membrane receptor sites include: (1) lipids, (2) thrombin, (3)
collagen, (4) adenosine diphosphate (5) epinephrine, and (6) thromboxane A2.
The release of the chemicals enhance the hemostasis process.
Comment: During the adhesion sequence, there is a simultaneous
releasing of platelet factors. See Objective #12.
11
DISCUSS THE PRIMARY AGGREGATION PHASE OF PLATELET PLUG
FORMATION
The attachment of platelets to each other is called aggregation. This
attachment phenomenon appears to occur in two stages. The first or primary
stage is the loose conglomeration of platelets that occurs before the secretory
phase begins. This primary stage is unstable can be reversed if the stimulus is
weak that initiated the adhesion process. If the aggregation phenomenon is
intense and aggressive, the release of chemicals will initiate the secondary
phase, which is stable and cannot be reversed.
12
DISCUSS THE SECRETION PHASE OF PLATELET PLUG FORMATION.
This is also called the release reaction step. This phase may begin immediately
with an intense adhesion phenomenon. There is a release phenomenon by the
dense bodies/granules in which serotonin, ADP, and calcium are initially
released. The Open Canalicular (OCS) begins to move calcium ions to the
outside to increase the extracellular Ca++ levels. Alpha granules also release
their substances. The secretion phase helps to convert the aggregating
platelets into a mass of degenerated platelets without membranes. This
transformation phenomenon is called viscous metamorphosis. ADP binds with
the platelet receptors mobilizes the fibrinogen binding sites.
13
LIST SIX SECRETORY PRODUCTS RELEASED BY DENSE GRANULES OF
PLATELETS AND IDENTIFY WHAT ROLE THEY HAVE IN THE CLOTTING
PROCESS.
[1] Calcium: activates Ca++ sensitive phospholipases which drive reactions to
form thromboxane A2 from arachidonic acid.
[2] ADP: bind to platelet membrane receptors to activate fibrinogen binding
sites.
[3] Serotonin: a vasoactive amine causing vascular constriction.
[4] Epinephrine: an amine active vasoconstrictor.
[5] ATP: energy molecule to facilitate platelet activation.
[6] Magnesium: cofactor for ADP and ATP.
14
LIST SEVEN SECRETORY PRODUCTS RELEASED BY ALPHA GRANULES OF
PLATELETS AND IDENTIFY THEIR ROLE IN THE CLOTTING PROCESS.
[1] Fibronectin: a protein with adhesive properties to bind platelets to each
other.
[2] Thrombospondin (TSP): a protein with adhesive properties to bind
platelets to each other.
[3] Fibrinogen: a clotting protein to form a bridge between platelets and
endothelium.
[4] Platelet Derived Growth Factor (PDGF): a mitogen that promotes cellular
proliferation in the endothelial cells of the damaged vessel.
[5] von Willebrand factor: facilitates attachment of platelets to endothelial
cells.
[6] -thromboglobulin: a globulin that has weak anti-heparin activity.
[7] 2 - antiplasmin: an enzyme that inactivates plasmin and prevents clot
lysis.
15 DISCUSS THE SECONDARY AGGREGATION STEP IN THE FORMATION OF
THE PLATELET PLUG.
At this step of the platelet plug formation phenomenon, specific platelet
receptors for the clotting proteins are now exposed. The platelet plug, up to
this point of it development, is unstable and can be dislodged and washed
away. The secondary step is characterized by stabilization and the firm fixation
of the platelet plug to the injury site. This is accomplished by (1) formation of
fibrin, (2) continued contraction of the platelet mass, which extrudes serum,
(3) binding of coagulation factors to the platelet receptor sites, (4)
entrapment of RBCs in the platelet-fibrin clot, and (5) hardening of the fibrin
polymer by fibrin-stabilizing factor (XIII).
The secondary aggregation step is also characterized by additional
degranulation within the platelets, releasing more substances to drive the
platelet plug process into it irreversible phase.
16
DESCRIBE HOW ASPIRIN AFFECTS PLATELET ACTIVITY.
[1] It inhibits the production of platelet cyclo-oxygenase, an enzyme that is

required for the production of prostaglandins and thromboxanes that have
diverse functions that include vasoconstriction of blood vessels, platelet
aggregation, and blood pressure regulation.
A. Cyclo-oxygenase (also written as cycloxygenase) is required to
convert arachidonic acid to PGG2 and PGH2. These intermediates can
enter one of two pathways. If the endothelial surfaces are stabilized
and become relatively intact, prostacyclin synthetase is released,
producing prostacyclin (PGI2), which causes inhibition of platelet
aggregation and acts as a vasodilator. Prostacyclin will spontaneously
degrade to the inactive substance, 6-keto-PGF1a.
[2] If the endothelium is not stabilized then the enzyme thromboxane
synthetase is released, causing the synthesis of thromboxane A2 (Tx A2), which
causes increased platelet aggregation and increased vasoconstriction.
[3] Cyclo-oxygenase is produced in both endothelial cells and platelets.
Increased and sustained doses of aspirin will affect this enzyme and decreases
the potential for thrombosis. Aspirin inactivates cyclo-oxygenase by causing an
irreversible acetylation by the introduction of a CH3 - CHO - group into the
enzyme molecule.
[4] If a patient is taking aspirin as a therapeutic preventative measure, they

must discontinue for eight days before reliable bleeding times can be obtained.
17
BRIEFLY DESCRIBE HOW CLOT RETRACTION OCCURS.
Platelets contain in their cytoplasm, structural proteins that include actin and
myosin. In clot retraction, calcium and ATP stimulated actin and myosin
molecules contract to reduce the size of the plug and extrude plasma. This
contraction phenomenon is dependent upon the presence of fibrinogen
receptors (glycoprotein IIb/IIIa complex) that are occupied by fibrinogen,
fibrin, or fibronectin. NOTE: If these receptors are missing from the platelet,
the platelet(s) will not contract. This condition is known as thrombasthenia.
18 GIVE ONE EXAMPLE OF HOW PLATELETS CONTRIBUTE TO THE FIBRINFORMING SYSTEM.
Platelets produce phospholipid platelet factor 3 (PF-3) at a hidden site on the
membrane. When this factor is exposed, it becomes a catalytic molecule that
can activate selected clotting factors such as factors IX, X, and II.
19 EXPLAIN WHAT IS MEANT BY THE TERM, FIBRIN FORMING SYSTEM.
It is the interaction of the protein clotting factors to form a fibrin clot which
will help to stabilize the platelet plug.
20 WHAT IS G-PROTEIN?
G protein (also called GTP binding protein) is a transducing protein in the
membrane of a cell (or the platelet). This protein is a complex molecule made
up of three different subunits designated as , , and . The combination of
subunits determines if it has an inhibitory or activating action. If the G protein
is activating, it will act upon an enzyme, activating it and a product produced.
A substance may be released or prevented from being released or allosteric
changes may occur in a tubular system
21
DISCUSS HOW PLATELET ACTIVATION MIGHT BE REVERSED.
Platelet inhibition will not be initiated if alpha granule release has occurred. If
prostacyclin (PGI2) is released by healthy, intact epithelial cells; cAMP (3',5'cyclic-adenosine monophosphate) levels are increased and calcium
sequestration occurs. This acts as a signal in the dense tubular system of the
platelet that will cause certain protein receptors to become phosphorylated
and assume a negative charge. This attracts the cellular Ca++ from the cellular
spaces and reducing it availability to the activation process. G protein, found in
the platelet, is activated by cAMP. G-protein causes the activation process in
the dense tubular system.
22 EXPLAIN THE MEANING OF THE TERMS, PRIMARY HEMOSTASIS AND

SECONDARY HEMOSTASIS?
Primary hemostasis refers to the formation of the platelet plug. Secondary
hemostasis refers to the formation of the fibrin clot.
23 LIST FOURTEEN COAGULATION FACTORS, COMMON NAMES,
ABBREVIATIONS AND/OR SYNONYMS.
factor
common name (with known synonyms)
I
Fibrinogen
II
Prothrombin
III
Tissue Thromboplastin; Tissue Factor
IV
Ionized Calcium (Ca++)
V
Proaccelerin, Labile Factor, Prothrombin Accelerator,
Accelerator
Globulin (AcG), Thrombogen
VI
This designation is now unassigned. It was formerly listed as a clotting
factor, it is now known that it was an impure derivative of Factor V. It is now a
discarded term and textbooks do not list this factor nor indicate it as being
unassigned.
VII
Stable Factor, Serum Prothrombin Conversion Accelerator (SPCA),
Proconvertin
VIII Antihemophilic Factor (AHF), Antihemophilic Factor A, Antihemophilic
Globulin (AHG), Platelet Cofactor 1.
IX
Christmas Factor,, Antihemophilic Factor B, Prothrombin II, Platelet
Cofactor 2, Plasma Thromboplastin Component (PTC).
X
Stuart-Prower Factor, Stuart Factor, Autoprothrombin III, thrombokinase.
XI
Plasma Thromboplastin Antecedent (PTA),, Antihemophilic Factor C,
Prothrombin Antecedent
XII
Hageman Factor, Contact Factor, Glass Factor
XIII
Fibrin-Stabilizing Factor (FSF), Transglutaminase, Fibrinoligase, LakiLorand Factor (LLF), Fibrinase, Plasma Transglutaminase
Fitzgerald High-Molecular-Weight Kininogen (HMWK), Flaujeac Factor,
Contact Activation Factor, Williams Factor, Reid Factor, Washington Factor
Fletcher Prekallikrein (PK)
24
DESCRIBE HOW THE COAGULATION FACTORS MAY BE GROUPED
ACCORDING TO THEIR PHYSICAL PROPERTIES.
Coagulation factors may be divided into (1) contact proteins, (2) prothrombin
proteins, and (3) fibrinogen proteins.
There are four contact type of coagulation factors. These are plasma
thromboplastin antecedent (XI), Hageman factor (XII), pre-kallikrein (PK), and
high-molecular-weight kininogen (HMWK). These are involved in the initial
activation of the intrinsic coagulation pathway. They require a negatively

charged surface to activate. Only Factor XI appears to have an essential role in
the clotting scheme.
The four prothrombin types of coagulation factors are prothrombin (II), stable
factor (VII), Christmas factor (IX), and Stuart-Prower Factor (X). These are
low-molecular weight factors and they require vitamin K for synthesis. These
factors contain gamma carboxyglutamic acid that is necessary for binding
calcium ions. The -carboxyglutamic acid component (with the calcium ions)
enables the molecule to be attracted to the acidic phospholipid surface of the
platelet to take part in the clotting process. These factors can be precipitated
and removed by barium sulfate or aluminum hydroxide. These factors are
produced in the liver. Other prothrombin-type proteins known to have an effect
in coagulation are protein C and protein S (See Objective 56 and Objective 57).
NOTE: If vitamin K is absent, the clotting proteins are synthesized but without
the functional carboxyl groups to bind calcium. These proteins may be
referred to as Protein Induced by Vitamin K absence / antagonist (PIVKA).
The four fibrinogen types of coagulation factors are [1] fibrinogen, (2)
proaccelerin (V), [3] antihemophilic factor (VIII), and [4] fibrin-stabilizing
factor (XIII). These are present in normal and absorbed plasma. They are not
found in serum because they are consumed in the clotting process (formation
of fibrin). Factors V and VIII are the least stable of the clotting factors because
of their labile tendency to degradation and denaturation.
Labile describes the tendency to be unstable; unsteady, not fixed. It denotes:

1. An adaptability to alteration or modification, i.e., relatively easily changed or
rearranged.
2. Certain constituents of serum affected by increases in heat.
3. Easily removable; e.g., a labile hydrogen.
25 LIST THE COAGULATION FACTORS DESCRIBED AS SERINE PROTEASES

Hageman factor (XII)
Fletcher factor
Prothrombin (II)
Stable Factor (VII)
Plasma Thromboplastin Antecedent (XI)

Christmas Factor (IX)
Protein C
NOTE
Serine proteases require an active serine molecule in the catalytic site. The
target site of serine proteases are specific peptide bonds in protein molecules
and in many cases their target molecule is another molecule containing serine.
Protein activation by these proteases tends to be limited to the hydrolysis of
one or two peptide bonds.
26 LIST THE KNOWN CLOTTING FACTORS AND DESCRIBE THE FOLLOWING

FEATURES FOR EACH OF THE FACTORS: [1] BLOOD CONCENTRATION, [2]
HALF-LIFE IN HOURS, [3] VITAMIN K DEPENDENCY, [4] COAGULATION
PROTEIN GROUP, [5] TYPE OF PROTEIN, [6] SITE OF PRODUCTION, [7]
CLOTTING PATHWAY INTERACTION, [8] STORAGE , AND [9] BIOCHEMICAL
FUNCTION.
A.
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
Fibrinogen (I)
blood concentration 200 to 400 mg/dL
half-life, hours 90 to 120 hours
vitamin K dependency: not dependent
coagulation protein group: fibrinogen
type of protein: glycoprotein
site of production: liver
clotting pathways: intrinsic, extrinsic, and common
storage: stable
biochemical function: substrate
B. Prothrombin (II)
[1] blood concentration: 10 mg/dL
[2] half-life, hours: 60 hours to 100 hours
[3] vitamin K dependency: yes
[4] coagulation protein group: prothrombin
[5] type of protein: 2 - globulin
[6] site of production: liver
[7] clotting pathways: intrinsic, extrinsic, and common
[8] storage: stable
[9] biochemical function: serine protease
C. Tissue Thromboplastin (III)
[1] blood concentration: none
[2] half-life, hours: not known
[3] vitamin K dependency: no
[4] coagulation protein group: none
[5] type of protein: lipoprotein
[6] site of production: all tissues, it is highest in the brain, liver, lung,
placenta.
[7] clotting pathways: extrinsic
[8] storage: stable
[9] biochemical function: cofactor
D.
[1]
[2]
[3]
[4]
Ionized Calcium (IV)

blood concentration: 4.8 to 4.92 mg/dL (2.24 to 2.46 mEq/L)
half-life, hours: nor applicable
coagulation protein group: none
[5]
[6]
[7]
[8]
[9]
type of protein: not a protein

site of production: absorbed across the intestinal wall
clotting pathways: intrinsic, extrinsic, common
storage: stable
biochemical function: cofactor
Ionized calcium is different from total calcium which makes up 8.4 to 10.2
mg/dL. 45% is protein bound, 45% is ionized, and 10% is ligand (lactate, citrate,
phosphate, and bicarbonate) bound
E.
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
Proaccelerin (V)
blood concentration: 5 to 12 g/mL
half-life, hours: 12 to 36
storage: labile
F.
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
Proconvertin (VII)
half-life, hours: 5 to 8 hours
vitamin K dependency: yes
coagulation protein group: prothrombin
clotting pathways: extrinsic
storage: stable
biochemical function: serine protease
G.
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
Antihemophilic Factor (VIII)

type of protein: multi-protein complex
clotting pathways: intrinsic
storage: labile
H. Christmas Factor (IX)

[1] blood concentration: 3 to 4 g/mL
[2] half-life, hours: 20 to 72
[3]
[4]
[5]
[6]
[7]
[8]
[9]

type of protein: dipeptide chain linked by disulfide bonds
storage: stable
I.
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
Stuart-Prower Factor (X)

storage: stable
J.
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
Plasma Thromboplastin Antecedent (XI)

coagulation protein group: contact
type of protein: two identical polypeptide chains lined by a disulfide bond
storage: stable
K.
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
Hageman Factor (XII)

type of protein: glycoprotein, specifically a 2 - globulin
storage: stable
L.
[1]
[2]
[3]
[4]
Fibrin Stabilizing Factor (XIII)

[5]
[6]
[7]
[8]
[9]
type of protein: double chain 2 - globulin

storage: stable
M.
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
Fletcher Factor
half-life, hours: approximately 35
type of protein: gamma globulin
storage: stable
N.
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
Fitzgerald Factor
half-life, hours: approximately 156
storage: stable
NOTE THAT ONLY FACTORS V AND VIII ARE LABILE.

27
DISCUSS AND / OR ILLUSTRATE THE EXTRINSIC COAGULATION PATHWAY.
The extrinsic coagulation pathway is an alternate pathway describing how

Factor X can be activated and requires Factor III (tissue thromboplastin). This
pathway is initiated external to the vascular system. Tissue thromboplastin is
located outside the vascular system and is a membrane protein. When tissue
trauma occurs, it is released and begins the cascade process. Proconvertin (VII)
is unique to this system and will bind with tissue thromboplastin in the
presence of calcium . The extrinsic cascade coagulation sequence is as follows:
28
DISCUSS AND / OR ILLUSTRATE THE INTRINSIC COAGULATION PATHWAY.
This is a sequence of initiating coagulation events that occurs within the

vascular system (hence the reason for its intrinsic designation). This reaction
sequence begins with the auto-inactivation of Hageman Factor (XII). Factor XII
requires exposure to a foreign substance such as collagen, phospholipids, or
activated Fletcher factor. Actually the role of Fletcher and Fitzgerald factors
are to enhance and amplify the contact factors role. There are six factors
involved in this pathway: factors VIII, IX, XI, XII, Fletcher, and Fitzgerald.
Factor X is a part of all three pathways because of its unique position in the
cascade.
To initiate the cascade scheme there must be the development of three
enzyme complexes on the platelet membrane. A complex of VIIa and tissue
factor + VIIIa and IXa and Ca++ and PF3 + Xa and Va and Ca++ and PF3.
Intrinsic Cascade Schematic
28
DISCUSS AND / OR ILLUSTRATE THE COMMON COAGULATION PATHWAY.
The coagulation phenomenon of the common pathway begins with Stuart Factor
(X). Factor X is activated by the extrinsic or intrinsic system or both. The two
principle reactions in this pathway is the conversion of prothrombin to
thrombin and fibrinogen to the fibrin monomer.
29
EXPLAIN THE TENASE COMPLEX IN THE COAGULATION PATHWAY.
This is a reaction step that occurs on the platelet membrane as a part of the
intrinsic pathway. It is the formation of an enzyme complex between Factor IX
and VIII:C, Ca++, and platelet factor 3 (PF3). This enzyme complex, as it sits on
the platelet membrane will activate Factor X. The complex is called the tenase
complex because of the conversion of the inactive factor X to its active form.
The surface of the platelet provides a protective environment for the
coagulation reactions from the interferences of opposing molecules.
30
ILLUSTRATE HOW THE VARIOUS COAGULATION PATHWAYS INTERACT.
Refer to the next three pages designated as Coagulation Pathways. These pages
may be attached to form a three-page fold out.
31
DISCUSS THE COMPLEX NATURE OF COAGULATION FACTOR VIII.
Factor VIII is a complex of several components and constitutes the largest

molecule in the coagulation scheme. The two principle subunits of Factor VIII
are:
[1] Subunit I, which is a procoagulant and may be written as VIII:C.
A. This part of the molecule is the antihemophilic factor.
B. Certain receptors of VIII:C bind to Ca++ and then binding to the platelet
occurs.
C. The coagulant properties are in this subunit.
[2] Subunit II, which is that portion of factor VIII that bonds with von
Willebrand Factor (vWF) and may be designated as the VIII-vWF complex. The
complex forms the functional molecule.
[3] Both subunits I and II form the basic molecule. The acronym VIIIAg
designates that part of the basic molecule that is antigenic.
Factor VIII:C production occurs primarily in the hepatocytes and is controlled
by genes in the X-chromosome. It is somewhat unstable and has a short half life
(8 to 12 hours). When it complexes with vWF, it increases it stability several
fold. It is this portion of Factor VIII that is under produced or not at all in
hemophilia. Each part of factor VIII has its own biological and immunological
properties. Note that some of Factor VIII circulates free but will not be
functional.
32 DISCUSS von WILLEBRAND FACTOR (vWF) AND ITS ROLE IN
COAGULATION.
vWF is a glycoprotein and is thought to be synthesized in the endoplasmic
reticulum of endothelial cells (the Weibel-Palade bodies) and megakaryocytes.
This factor is released and absorbed onto the surface of platelets and stored in
the alpha granules of the platelet. It is also absorbed onto the endothelial
lining of the vascular system, where it is stored in granules called WeibelPalade bodies. vWF is a complex molecule, associated with factor VIII. It is
described as a large, adhesive multimeric glycoprotein with a molecular weight
(Daltons) from 600,000 to 20 X 106. vWF exists in several entities. The low
molecular weight form (220,000 Daltons) is a monomer. It has capability to
form a type of polymer composed of a variable number of subunits (dimers,
trimers, tetramers, and multimers), giving rise to the higher molecular weight
molecules which supports vWF activity. When injury occurs and platelet
activation begins, the vWF that is bound to the platelets glycoprotein Ib and
glycoprotein IIb/IIIa receptors, will bind by another receptor site to the
exposed endothelial collagen and/or heparin, forming a bridge. vWF is
essential for the platelet to adhere to the collagen fibers. The vWF subunit
contains (1) antigenic determinants, (2) ristocetin receptors, and (3)
collagen/platelet receptors. Note: A prolonged bleeding time may
indicate a vWF deficiency as can an abnormal APTT and ristocetin
platelet aggregation test. This can be substantiated by a vWF assay.
Acronyms used with the vWF are as follows:
vWF refers to the basic molecule regardless of whether it is a dimer, trimer or
multimer.
vWF:Ag (called von Willebrands antigen) is the antigenic portion of the basic
molecule without regard to its molecular weight and ability to function. It as
been recommended that this acronym be discontinued or interpreted with

caution.
vWF:Co represents the reactive portion of the basic molecule.
The following facts pertain to the vWF molecule:
[1] vWF makes up 90% of the VIII:vWF complex
[2] vWF appears to have no coagulant properties, but acts as a stabilizer for
factor VIII.
[3] vWF gives factor VIII distinctive antigenic characteristics.
[4] vWF provides factor VIII with the binding sites to attach to the platelet
and injury site. The large size of this complex enable binding the platelet to
the site of injury.
[5] Genes in chromosome 12 control production of vWF.
[6] vWF formed in the endothelial cells is deposited throughout the
subepithelium.
[7] vWF formed in the megakaryocytes is deposited in the -granules of the
platelet. This vWF is not complexed to factor VIII and is of a larger molecular
weight.
33 LIST SIX ROUTINE SCREENING TESTS THAT ARE USED TO DETECT THE
PRESENCE OF A COAGULATION PROBLEM.
[1] Platelet count, [2] bleeding time, [3] prothrombin time, [4] activated
partial thromboplastin time, [5] thrombin time, [6] tourniquet test. NOTE:
This is a generalization and may vary from lab to lab.
34 DISCUSS SPECIMEN COLLECTION REQUIREMENTS OF COAGULATION
TESTING.
The Venipuncture: This must be done correctly and trauma free to eliminate
any possible contamination of the blood with tissue fluids. The vacutainer
system is satisfactory, collecting two or three tubes. The first tube will be
discarded upon returning to the laboratory. If special coagulation procedures
are required, the two syringe collection technique may be required.
Two Syringe Method
Use sterile plastic syringes. Draw approximately 2.0 mL of blood into the first
syringe, then disconnect this syringe from the needle and attach the second
syringe. Proceed with the venipuncture and transfer the contents of the second
syringe into the proper coagulation tube. The first syringe may be discarded
when you have returned to the laboratory.
Phlebotomy: When drawing blood from the patient, release the tourniquet
immediately after blood enters the first tube. Allow the tube to fill until no
vacuum remains. It is important to maintain the correct blood to anticoagulant
ratio. As long as the blood fills the tube within 10% of the tubes expected
volume, the ratio may be considered to be correct. If a greater or lesser
amount of blood fills the tube, incorrect coagulation results will be obtained.
The Tubes: Vacutainer tubes need to have a non-contact surface, an inert
material that should not react with the coagulation factors. Vacutainer tubes
have a siliconized surface. Glass or soda line can react with Hageman factor
(XII) to initiate clotting and consumption of clotting factors which will
invalidate the test.
Anticoagulant: The anticoagulant of choice is 0.109 M (3.2%) sodium citrate.
Buffered or non-buffered citrate may be used. 0.129 M (3.8%) sodium citrate
has been used in the coagulation studies but is not a recommended
concentration. Buffered citrate is preferred because the pH of blood is
stabilized which give more consistent/reliable results. The ratio of
anticoagulant to blood is one part anticoagulant to nine parts of blood. This is a
1 to 10 dilution. Many labs use a 5.0 mL vacutainer that contains 0.5 mLs of
sodium citrate. This tube will draw 4.5 mLs of blood for a 1 to 10 dilution.
Note: If the sodium citrate anticoagulant is buffered, it will contain citric
acid. The sodium citrate used to make up the anticoagulant may be trisodium
citrate dihydrate.
Caution: Do not use capillary specimens for coagulation procedures. It
is next to impossible to collect a capillary specimen without trauma or
contamination with tissue fluid.
35 DESCRIBE THE BLOOD COLLECTION STRATEGY FOR A PATIENT WITH A
HEMATOCRIT OF 55% OR HIGHER.
Blood specimens that are characterized by elevated hematocrits have a smaller
volume of plasma. Remember that sodium citrate has the potential to
concentrate in the plasma creating an excess that has the potential to modify
test results. The problem lies in the reagents used in the screening tests that
contain calcium. At these concentrations, sodium citrate may compete for
calcium ion introduced in the screening test and produce a false result. This
will prolong the clotting time. The solution is to remove a portion of the
sodium citrate in the collection tube to maintain a proper anticoagulant to
plasma ratio. In the event that the patient has an elevated hematocrit, you can
correct the problem by following the outlined steps:
[1] determine the hematocrit.
[2] use the following formula to correct for the amount of citrate needed.
A. Divide (100 - hematocrit) by (595 - hematocrit) to obtain the correction
factor.
B. Multiply the correction factor by the mLs of whole blood used. The volume
of blood to be treated with anticoagulant will be 4.5 mLs or 9.0 mLs.
[3] Example: If a patient has a hematocrit of 58% . . . .
A. 100 - 58 = 42 and 595 - 58 = 537 therefore 42 divided by 537 = 0.0782

B. multiply 0.0782 times 4.5 (mLs of blood to be collected) = .352 mLs.
C. Place 0.35 of .109 M sodium citrate in a test tube and add 4.5 mLs patients
blood.
Nomograms are available to calculate the amount of anticoagulant required to
maintain a 1:10 anticoagulant ratio in the blood. Refer to the following graph
as an example.
36
LIST FIVE STEPS FOR HANDLING THE PATIENTS SPECIMEN AFTER
COLLECTION FOR COAGULATION TESTING.
STEP 1: After returning to the laboratory and before and after centrifugation,
examine the specimen for clots. NOTE: Blood is centrifuged with the cap
or stopper on. It is not removed. After removing the plasma for
coagulation testing, it proper to re-examine the red cell layer for clots.
If the plasma appears hemolyzed, it is indicative of a traumatic
venipuncture and should not be used for coagulation testing. Also if the
plasma appears lipemic or icteric, it should not be used. If a clot or clots
are found in any of the examination stages, the specimen is not suitable for
testing. A new specimen must be collected. To avoid the possibility of the clot
formation, collect the specimen quickly via a flawless venipuncture and mix
the tube of blood gently but thoroughly and immediately after collecting.
STEP 2: Complete the coagulation testing within 30 minutes to 2 hours. NOTE:
Some procedures may be performed within a four hour time limit. Such
time limits are specified in a particular laboratorys procedure manual.
Remember that once the blood is removed from its natural environment,
changes will begin to take place. Specimens may become deficient in
coagulation factors over time. Factors V and VIII are designated as labile
factors because they are very unstable and will quickly decrease during
storage. If the specimen has to be retained for an extended period of time, it
should be stored on ice or held in the refrigerator at 4 oC. If prolonged storage
is required, store the plasma at -20 oC. Freezing should be conducted rapidly to
prevent denaturation of the coagulation proteins. To rapidly freeze specimens,
use temperatures of -40 oC to -80 oC. Liquid nitrogen will accomplish this
nicely. When you are ready to test the patients specimen, rapidly thaw the
specimen to 37 oC. Caution: Once plasma has been thawed, it cannot be refrozen.
STEP 3: All testing equipment that will come in contact with the test plasma
must have smooth and non-reactive surfaces, designated as non-contact or
siliconized.
STEP 4: Keep the test plasma stoppered. Leaving the tube un-stoppered after
centrifugation will result in gaseous exchange with the atmosphere. CO2 tends
to evaporate from the plasma in an un-stoppered tube. Such CO 2 loss causes pH
changes that are detrimental to coagulation activity.
STEP 5: The plasma must be platelet poor. Such plasma is designated as
platelet poor plasma (PPP). A PPP will have a platelet count of <15,000 L. The
blood should be centrifuged at 3000 rpm for 10 minutes. After the centrifuge
stops, quickly remove the plasma to a separate tube and stopper.
37
DISCUSS THE IMPORTANCE OF HEATING IN COAGULATION TESTING.
Most coagulation tests are conducted at 37 oC. It takes slightly longer for
plasma to come to temperature if the lab uses a dry block incubator. Testing
strategy requires setting up the test so that the patient specimen and reagents
will come to the proper temperature simultaneously. It takes about three
minutes to warm up plasma for testing. Do not prewarm plasma beyond 10
minutes before testing. Overheating and prolonged heating is to be avoided to
prevent denaturing the coagulating factors and causing prolonged tests results.
Denaturation can begin at 45 0C. Incubators (dry block or water) must hold
temperatures within 0.5 0C.
38
DISCUSS THE TESTING STRATEGY FOR DETERMINING THE END POINT
OF COAGULATION.
Most tests are designed to detect the time required for the formation of a
fibrin clot. Some tests may consist of combining calcium, thromboplastin (with
or without an activator) and citrated plasma. The fibrin clot can be detected
either visually, electro-mechanically, or by change in the optical density.
Originally the tilt tube or loop method were employed to detect the clot. The
tilt tube required continuous tilting of the tube back and forth until the formed
clot became visible. The loop method required the use of a wire that was
passed through the mixture (about two sweeps per second) until a clot
attached to the wire. The next step in the evolution of clot detection utilized a
set of electrodes that would be submerged in the reaction mixture and sweep
back and forth until a clot formed. The developed clot forms an electrical
circuit that will stop the timer. The next generation of automated coagulation
devices uses optical density (spectrophotometry). In this case the formation of
the fibrin clot causes a change in the optical density of the plasma, also
resulting in the stopping the timer.
39
DESCRIBE IN SIMPLE TERMS THE PROTHROMBIN TIME PROCEDURE.
The prothrombin time (PT) requires [1] the placement of a designated amount
of thromboplastin reagent in the reaction well or tube, [2] allowing the plasma
temperature to adjust to 37 oC, and [3] add a designated amount of citrated
platelet poor plasma (PPP). When all reagents have been added allow mixing
to take place and record the time that the fibrin clot forms. A normal PT test
will be in the range of 10 to 13 seconds.
40
BRIEFLY DESCRIBE HOW TO EVALUATE A PROTHROMBIN TIME TEST.
If the time extends 2-3 seconds beyond the normal range, this may indicate a
problem. If the prothrombin time (PT) procedure is performed in duplicate, the
second test must be within 10% of the first test. If factors is IX, VII, and/or X
are 20% to 65% of normal, the PT many be prolonged by 1 to 3 seconds. The PT
is prolonged when factor II is <10% of normal and fibrinogen is <100 mg/dL. If
the PT patients test result is three times that of normal, the patient is at risk
for hemorrhage and the physician may administer an antidote. If a coagulation
scheme is reviewed, it would indicate that the PT test would be prolonged if
there was a deficiency in factors I, II, V, VII, VIII, IX, and X. In reality the PT
test is most sensitive to a factor VII deficiency and moderately sensitive to
deficiencies in factors V and X. It is sensitive to a marked deficiency in either
Factor I and II. It is totally insensitive to deficiencies in factors VIII and IX.
NOTE
The plasma aliquot must be incubated for at least three minutes, but no longer
than ten minutes. Factors V and VIII are labile and will deteriorate after ten
minutes of incubation time. Evaporation also occurs, which affects the quality
of the test. Long incubation times results in evaporation or reagents and
adversely affects the PT. The net effect is that the PT will be falsely prolonged.
When setting up reference intervals, one laboratory may establish a normal
control range of 12 to 14 seconds and another laboratory use 11 to 13 seconds
as its normal range. Reference ranges can be affected by the patient
population, type of thromboplastin, type of instrumentation, and the pH and
purity of the distilled water. Each laboratory should establish its own normal
range annually using a minimum of 20 healthy individuals of both sexes.
41 DISCUSS AND/OR CALCULATE THE INTERNATIONAL NORMALIZED RATIO
(INR).
The International Normalized Ratio (INR) is an attempt to standardize the
thromboplastin reagents of the different reagent manufacturers to that of the
international reference thromboplastin [World Health Organization (WHO)
human brain thromboplastin]. This process has developed because different
thromboplastins do not have the same sensitivity. Manufacturers calibrate each
lot of their PT thromboplastin against the standard WHO thromboplastin
reagent. The reagent manufacturers will then calculate an international
sensitivity index (ISI) number for their reagent. This ISI number can be entered
into the automated coagulation instrument and a corrected calculation can be
made for each patient. Programmable hand-held calculators can formulate the
INR if the ISI is entered. Manufacturers provide tables for their reagents and
the PT ratio and ISI can be looked up and reported. The INR is the patients
prothrombin time ratio to the power equal to the ISI. The following formula
shows how to calculate the INR.
INR = (PTpatient / PTnormal)ISI
The following problem is provided as an example. Assume that the patients PT
is 16 seconds, the normal control is 12 seconds, and the ISI = 2.0. The problem
will set up as follows:
INR = (16 seconds / 12 seconds)2.0
INR = (1.33)2.0
INR = 1.768
The value of this INR number is seen when this same patient is tested by
another laboratory using different prothrombin testing reagents. If the second
lab reported out the same patient with a PT of 17 seconds and its normal
control was 10 seconds, is this PT results the same or different than the first
lab?
Assume that the ISI number assigned to their test reagents is 1.9. The
calculated INR is 2.7. The INR numbers between the two labs is different and so
are the testing procedures. If a third lab performed the prothrombin test on
the same patient with the following results: PT = 27 seconds, the normal
control = 12 seconds, and their ISN number is 1.2. The calculated INR becomes
2.7. The identical INR numbers means that there is no difference in the PT
tests between the last two labs.
The INR appears to be useful for patients who are on long term
oral anticoagulants and have been stabilized. The INR is not
recommended for patients who are beginning oral anticoagulant
therapy, the evaluation the coagulation status of presurgical
patients, nor for a patient with a liver disease.
Physicians used NR results to determine the level of coumadin therapy usually
after the patient has been on anticoagulant therapy for at least two weeks.
The dosages are adjusted to keep the patients prothrombin time to a INR value
of 2.0 to 3.0 or possibly 3.5 if the patient has a mechanical heart valve. INR
values greater than 4.0 tend to place the patient at risk of hemorrhaging.
42
DESCRIBE COMMERCIAL THROMBOPLASTIN.
Also known as tissue factor III, thromboplastin is an organic extract of rabbit

brain or lung tissue suspended in a buffered solution of citrate. This solution
may or may not have calcium added. If the ISI value of thromboplastin is close
to 1.0 then the thromboplastin is sensitive. The closer the ISI value is to 2.0 the
less sensitive the thromboplastin. Commercial thromboplastin is insensitive to
factor IX due to the concentration of reagents but if it is diluted, the sensitivity
to factor IX increases but the reagent becomes less sensitive to factors II, VII,
and X.
43 DESCRIBE WHAT FACTORS ARE EVALUATED IN THE FOLLOWING
COAGULATION SCREENING TESTS.
XIII
XII
XI
X
IX
VIII
VII
V
II
I
bleeding
clot
PT APTT
time
retraction
no
no
no
no
no
no
no
yes
no
no
no
yes
no
no
yes
yes
no
no
no
yes
no
no
no
yes
no
no
yes*
no
no
no
yes
yes
no
no
yes
yes
no
yes
yes
yes
TT
no
no
no
no
no
no
no
no
no
yes
urea
solubility
yes
no
no
no
no
no
no
no
no
no
Fletcher no
HMWK
no
Platelets
yes
*most sensitive
44
no
no
yes
no
no
no
yes
yes
no
no
no
no
no
no
no
DISCUSS THE PROTHROMBIN TIME (PT) AS A SCREENING PROCEDURE.
This test is also called pro-time. This is the most often ordered test to
monitor Coumadin oral anticoagulant therapy. This is the preferred screening
method for extrinsic pathway abnormalities, detecting deficiencies in factors
II, VII, IX, and X. If the factor concentration is decreased to 20% to 65% of
normal, the PT is prolonged for one to three seconds. In cases of
dysfibrinogenemia, where the fibrinogen concentration drops to 80 mg/dL,
the PT will be prolonged. If factor II drops to 10% of normal, the PT will be
prolonged. Other disorders that result in a prolonged PT are:
[1] dicumarol therapy
[6] heparin therapy
[2] factor V deficiency
[7] factor VII deficiency
[3] obstructive jaundice
[8] hemolytic disease of the newborn
[4] Vitamin K deficiency
[9] circulating anticoagulants
[5] factor X deficiency
[10] liver diseases
The PT is a comparison of the patients prothrombin time to that of a normal
control. If the normal control is 13 seconds and the patients PT is also 13
seconds, then there is 100% prothrombin activity. If the patients PT extends to
18 seconds, then the PT activity will be about 50%. If the PT should extend to
36 seconds, then the PT activity is less than 12%. The physicians goal in
coumadin therapy is to attain an INR value of 2.0 to 3.5 or a prothrombin time
that is 1.5 to 2 times that of the normal control plasma.. See Objective #41.
45 LIST TEN ERRORS THAT CAN OCCUR IN PERFORMING THE
PROTHROMBIN TIME.
Using the incorrect anticoagulant
Using the incorrect amount of anticoagulant
Using expired reagents.
Using an incorrect amount of blood to anticoagulant.
Using old or hemolyzed patients plasma
Using wet or dirty plastic ware or glassware in performing the tests.
Pipetting errors.
Failure to follow the manufacturers directions.
Changing from one manufacturer to another and not maintaining
careful quality control.
[10] Changing from one lot number to another without verifying the
control results.
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
46
BRIEFLY DISCUSS THE PRINCIPLE OF COUMADIN THERAPY.
Coumadin is a warfarin compound and competes with vitamin K in the liver

preventing synthesis of the vitamin-K dependent coagulation factors. Coumadin
is also known as Warfarin sodium, Coufarin, Panwarfin, Warfilone, and Warnerin
sodium. Coumadin is 4-hydroxycoumarin and inhibits the -carboxylation of
vitamin K dependent inactive clotting factors (II, VII, IX, and X). The absence of
these -carboxyglutamic acid units leaves these four clotting factors unable to
bind calcium ions. The clotting factors cannot congregate on the phospholipid
surface of the platelet nor to participate in clotting reactions.
When a patient is started on coumadin therapy, it become effective in about 10
hours. It takes about two weeks to stabilize the patient and for maximum
therapy effect. The physicians intent is to increase the PT to about 1.5 to 2.0
times that of normal. Each coumadin dose will be reflected from 36 to 48 hours
later. The PT measures VII activity, but the activity level of factors II, IX, and X
has the potential to affect the test.
Clinicians will prescribe oral anticoagulants to prolong the clotting time, inhibit
clotting, and facilitate clot lysis without placing the patient at risk of a
hemorrhage. Disorders in which coumadin may be prescribed are:
[1] intravascular clotting
[6] post-operative thrombophlebitis
[2] pulmonary embolism
[7] acute coronary thrombosis
[3] atrial fibrillation
[8] recurring idiopathic thrombophlebitis
[4] tissue heart valves
[9] recurring systemic embolism
[5] acute embolic and thrombotic occlusions of the peripheral arteries
Coumadin type drugs should not be prescribed for menstruating women,
pregnant females, nursing mothers, during postpartum, and following
cerebrovascular accidents.. The chief problem with coumadin therapy is
hemorrhagic accidents.
47 DESCRIBE THE THREE TYPES OF ORAL ANTICOAGULANTS USED BY
PHYSICIANS.
These are dicumarols, coumarins or coumadins, and indanediones (in-dan-e-dionez). Coumarins are most commonly used. The dicumarols are very slow
acting and indanediones have the most side effects. Because of the side effects
indanediones are avoided as therapy strategies. The antidote for coumarin is
vitamin K, but it may take up to three days to reach it full therapeutic effect.
Fresh frozen plasma can immediately correct an emergency bleeding due to
mis-dosage of coumarin. Caution: There is some degree of risk for
hepatitis or AIDS if using fresh frozen plasma.
48 LIST MEDICATIONS / DRUGS THAT ARE KNOWN AFFECT THE PATIENTS
RESPONSE TO COUMADIN THERAPY.
When a physician prescribes coumadin therapy, a review of what medications

the patient is currently taking should occur and a prescription written to
withdraw and/or add medications. Patients on coumadin therapy must be
monitored.
Drugs known to potentiate coumadin action are:
[1] ethyl alcohol
[8] allopurinol
[2] anabolic steroids
[9] aminoglycoside antibiotics
[3] tricyclic antidepressants [10] cephalosporins
[4] iodine based x-ray dyes
[11] heparin
[5] oral hypoglycemics
[12] penicillin
[6] sulfonamides
[13] choral hydrate
[7] glucagon
[14] thyroid hormone
Drugs known to depress coumadin effects are:
[1] barbiturates
[5] corticosteroids
[2] estrogens
[6] rifampin
[3] xanthines
[7] oral contraceptives
[4] carbamazepine
[8] phenobarbital
49 DESCRIBE THE BASIC PROCEDURE FOR THE ACTIVATED PARTIAL
THROMBOPLASTIN TIME (APTT) TEST.
The APTT procedure requires placing a designated volume of platelet poor
plasma (PPP) into a reaction well followed by a designated volume of
cephaloplastin reagent and allow the mixture to come to 37 oC. Three minutes
is ample time for step. The final step is to add a designated volume of calcium
chloride reagent. The ensuing reaction is watched and the time recorded when
the fibrin clot develops. This test may miss mild factor deficiencies. Normal
reaction time ranges from a low of 25 seconds to a high of 42 seconds.
Cephaloplastin is phospholipid substance obtained by
petroleum ether extraction of acetone dried brain tissue. The
APTT is called partial because it must have the assistance of
the plasma procoagulants (exceptions are factor VII and XIII). It
is considered to be a platelet phospholipoprotein substitute.
The testing procedure requires addition of an activator to assist
the activation of the contact factors.
50 DISCUSS THE PRINCIPLE OF THE ACTIVATED PARTIAL THROMBOPLASTIN
TEST.
The activated partial thromboplastin test (APTT) is also simply called the
partial thromboplastin time (PTT) test. This test evaluates the intrinsic clotting
system. The APTT test monitors Fletcher, Fitzgerald, XII, VIII, IX and XI
coagulation factors. This reagent contains a contact factor (Kaolin, ellagic acid,
or celite) which induces conformational changes in Hageman (XII) factor and
initiates the coagulation cascade mechanism. Most coagulation factor
deficiencies and circulating anticoagulants involve these factors, therefore
coagulation investigations should include the APTT as a testing strategy. APTT
reagent are calibrated to provide prolonged values when the patients plasma
has < 0.3 units/mL of factors VIII, IX, and XI. The APTT is also prolonged when
lupus anti-coagulant antibody is present. The APTT is insensitive to factors VII
and XIII.
51 LIST THE CIRCUMSTANCES IN WHICH A PHYSICIAN WOULD ORDER AN
ACTIVATED PARTIAL THROMBOPLASTIN TIME.
When the patient is suspected of having a hemorrhagic disorder or the
physician suspects a lupus anticoagulant antibody. The following are known to
cause prolonged APTTs:
[1] deficiencies of factors II, V, VIII, IX, X, XI, or XII.
[2] fibrinogen level <1.0 mg/dL
[3] anti-factor VIII
[4] fibrin degradation products (FDP)
[5] disseminated intravascular coagulation
[6] vitamin K deficiency (The APTT detects only Factors II, IX, and X)
[7] heparin therapy.
What kind of an APTT test result is the physician looking for in heparin therapy?
Since the normal range is from 25 to 42 seconds with a median time of 39
seconds, the physician is looking for a therapeutic time of about 58 seconds.
This is abut 1.5 times that of the median time.
52 DESCRIBE THE LUPUS ANTI-COAGULANT ANTIBODY.
This antibody is found in up to 10% of patients with systemic lupus
erythematosus (SLE), hence its name. It is also found in viral infections in
children and HIV. This antibody has an affinity for phospholipid, anti-factor VIII
antibody, and heparin. It will combine with the test reagents for APTT. The
lupus antibody may cause false positive serological tests for syphilis. Its
presence causes prolonged PT and APTT time results, but rarely causes
hemostatic problems. The antibody is more often associated with thrombosis
problems rather than those of bleeding. It is not known to activate clotting
factors. The antibody is either IgM or IgG or both. It binds to the phospholipids
blocking interaction in the coagulation scheme.
This antibody is difficult to identify in the laboratory. The following are a few
steps to follow in order to identify the antibody.
[1] Carefully collect the blood specimen and centrifuge to remove all
platelets because they will inactivate the antibody.
[2] Rule out heparin contamination. The thrombin time or reptilase test will
accomplish this.
[3] Mix one part of the patients plasma with one part of normal plasma. This
will correct the problem if it is a one factor deficiency. There will be no
correction for the presence of the antibody. Refer to Objectives 72 and 82.
[4] A Russell viper venom time (see Objective 81) or kaolin clotting time will
help to screen for the antibody. If these antibodies are present these tests will
be prolonged.
53
DESCRIBE HEPARIN AND ITS MODE OF ACTION.
Heparin (a sulfated polysaccharide) is a proteoglycan (a acidic

mucopolysaccharide) found inside mast cells and basophils, lung, liver, skin,
and in circulation. It is a heterogenous molecule that consist of several
components that can be separated by molecular size and weight. It is a unique
molecule containing many sulfate groups. One of its known functions is to
activate antithrombin III and another function is to interact with factor X,
prothrombin, and platelet aggregation. Caution: Heparin should not be
used to collect blood for platelet counts. It can be isolated from the liver,
lungs, and spleen. It is poorly absorbed from the GI tract and is usually
administered intravenously or subcutaneously as a sodium salt. Once in the
blood stream, its action is immediate. The activated clotting time and APTT are
used to monitor heparin therapy. The APTT test is sensitive to heparin and is
the test of choice. Physicians try to establish a therapeutic range with the APTT
test that is about 1.0 to 1.5 times that of the patients baseline or control
value. As a rule this puts the heparin concentration to about 0.3 to 0.5
units/mL. Side effects of heparin therapy are hemorrhage and development of
thrombocytopenia.
Heparin is thought to have binding sites for thrombin and antithrombin-III. By
complexing with thrombin and antithrombin-III, heparin can induce a
conformational change in antithrombin-III and facilitate the formation of
covalent, one-on-one thrombin-antithrombin-III complex, thus speeding up the
rate at which antithrombin III reacts with thrombin. One possible mode of
action is listed as follows:
54
DISCUSS THE ROLE OF PROTAMINE SULFATE IN COAGULATION.
It does not have a direct role. It is a simple protein that has been sulfated to
form an antagonist to heparin. One mg of protamine sulfate will neutralize 100
units of heparin. It is also a test procedure to evaluate the action of thrombin
on fibrinogen. See Objective 87.
55
DISCUSS ANTITHROMBIN-III AS A NATURAL INHIBITOR OF COAGULATION.
Antithrombin-III (AT-III) is a single-chain, 2 - globulin, produced in the liver,

and is a major inhibitor of coagulation. It is responsible for about 80% of
coagulation inactivation. Its an anti-serine protease, whose primary target is
inactivation of thrombin. It also inactivates any activated serine protease
clotting factor (IX, X, XI, and XII). Also inactivated are protein C and Fletcher
factor. AT-III contains an arginine residue that interacts with the active serine
molecule of the coagulation enzyme.
It is decreased in thrombotic disease, disseminated intravascular coagulation
(DIC) syndromes, cirrhosis of the liver, certain liver diseases, following surgery,
septicemia, and women on oral contraceptives or estrogen therapy. It is
assayed using electro-immunoassay, radial immunodiffusion, or enzyme-linked
immunosorbent assay. Normal ranges (17 to 30 mg/dL) are listed as low as 70%
and a high of 125%. Normal ranges are established by individual laboratories
and will vary from lab to lab. If AT-III levels drop to <60% that of normal, the
patient is at risk of a thrombosis.
56
DISCUSS PROTEIN C AS AN INHIBITOR.
Protein C (a glycoprotein) is vitamin K dependent and produced in the liver.

Plasma levels average from 3 to 5 mg/L. It is activated by thrombin and also a
potent inhibitor of Factors VIII and X. The activation of protein C is enhanced
and accelerated by the presence of thrombomodulin and Ca++. Endothelial cells
contain thrombomodulin molecules which interact with thrombin forming a 1:1
complex. The complex forms a modified thrombin molecule that is not free to
cleave fibrinogen, but is available to activate protein C. In this way thrombin

becomes an anticoagulant. Activated protein C (complexed with protein S)
destroys activated factor V, which limits the formation of thrombin. This
protein C activity make activated factor X more vulnerable to deactivation by
antithrombin-III. Protein C also enhances the release of tissue plasminogen
activator by inactivating a plasma inhibitor of tissue plasminogen activator.
57
DISCUSS THE ROLE OF PROTEIN S IN THE INHIBITION OF HEMOSTASIS.
Protein S is a vitamin K dependent glycoprotein and exists in plasma in two

forms. Forty percent of Protein S exists in the functional free form and the
remainder exists in a form complexed to C4b-binding protein (linked to the
compliment system) which is non functional. The amount of Protein S in plasma
is from 20 to 25 mg/dL. Normal laboratory ranges for Protein S ranges from 63%
to 160%. If protein S levels fall to 60%, then the patient is at risk for
thrombotic complications. Protein S is synthesized by hepatocytes and
megakaryocytes. This protein has a high affinity for phospholipid surfaces and
epithelial cells. This affinity promotes the binding of Protein C to such surfaces
and facilitates Protein C activation. Free protein S, in binding to Protein C,
forms a complex with Protein C. It is this complexed form of Protein C that acts
upon specific clotting factors.
58
DISCUSS 2 - MACROGLOBULIN AS A COAGULATION INHIBITOR.
2 - Macroglobulin is a glycoprotein found throughout the body. This protein will

form complexes with thrombin, plasmin, and Fletcher factor (kallikrein) to
inhibit their activities. The 2 - macroglobulin will bind slowly with thrombin,
then undergo cleavage, leaving the thrombin molecule less effective in its
proteolytic activity. It appears that the complexes formed by the 2 macroglobulin tend to be cleared from plasma by phagocytic cells of the
Mononuclear Phagocytic System (MPS), formerly the reticuloendothelial system
(RES). About 50% of the inhibition of Fletcher factor is conducted through the
macroglobulin. 2 - Macroglobulins are in the highest concentrations in infants,
small children, and pregnant women. It is reported that this glycoprotein can
form complexes with serine proteases.
59
BRIEFLY DESCRIBE THE PURPOSE OF C1-INACTIVATOR IN THE
INHIBITION OF HEMOSTASIS.
C1-Inhibitor (also called C1-inactivator) is a neuraminoglycoprotein. It is the
principle inactivator of activated factors XI and XII, plasmin, and activated
Fletcher factor (kallikrein).
60 BRIEFLY DESCRIBE THE PURPOSE OF 1 - ANTITRYPSIN IN THE
1 - Antitrypsin is a glycoprotein, which is thought to have a secondary role in

coagulation inactivation. It is considered to be a weak inhibitor of thrombin
and activated factors X and XI.
61 BRIEFLY DESCRIBE THE PURPOSE OF HEPARIN COFACTOR II IN THE
Heparin cofactor II is a glycoprotein synthesized in the liver. Its activity is
strongly augmented by heparin, indicating that it has limited activity by itself.
It is limited in its substrates, suppressing thrombin and chymotrypsin. There is
no known evidence of activity against activated factors IX, X, and XI.
62
DISCUSS THE ROLE OF VITAMIN K IN HEMOSTASIS.
Vitamin K is required by the hepatocytes to complete the alteration of factors

II, VII, IX, and X. It converts the glutamic acid residue in these factors to a carboxyglutamic acid residue that has the capability to bind calcium ions.
Vitamin K1, typical of plants, is found in green leafy vegetables. Bacterial flora
(Escherichia coli, Bacteroides fragilis, etc.) in the intestinal tract synthesizes
vitamin K2, a useable form characteristic to animals for absorption. If the
patient has mal-absorptive syndrome of the GI tract, vitamin K deficiency can
result. Other factors that lead to vitamin K deficiency are bile duct
obstruction, use of broad spectrum antibiotics, and sprue. Vitamin K is
depressed by the use of oral anticoagulants (coumadin) when given as an
anticoagulant therapy strategy. The therapeutic use of vitamin K can reverse
the deficiency within 24 hours.
Sprue is a tropical disease. Its actual cause is not known.
Symptoms include anemia, weight loss, and steatorrhea.
63 GIVE A BRIEF OVERVIEW OF FIBRINOLYSIS WITHIN THE CONTEXT OF
HEMOSTASIS.
The fibrinolytic system is activated when the coagulation system is activated.
The purpose of this mechanism is to digest (dissolve) fibrin clots as they are
formed to keep the vascular system free of emboli (clots). This process is
initiated when plasminogen is converted to plasmin. This proteolytic activity
produces fragments called fibrin degradation products (FDP). Plasmin is a
degrading enzyme which converts the fibrin clot into basic units called dimers.
Plasminogen activating factor (tPA) is attracted to the fibrin site where dimer
cross-linking occurs. This means that plasminogen will degrade at this site.
Cleavage of the fibrin clot produces the following products: first a YY/DXD
complex, second . . . DED and DY/YD complexes, and third . . . E-fragment, DD dimers, and DED complexes. The following degradation scheme illustrates the
degrading process.
64
DISCUSS THE ROLE OF PLASMINOGEN IN THE FIBRINOLYTIC SYSTEM.
Plasminogen is a single chain -globulin that circulates in the blood in an

inactive state. The plasma concentration ranges form 20 to 40 mg/dL. It is
produced in the liver. It is drawn into the clotting process in its inactive form
and becomes absorbed into the clot. Activators of plasminogen (t-PA) can be
found in the blood and most tissues. Active coagulation factor XII, Fletcher
(kallikrein), and Fitzgerald (HMWK) convert plasminogen to plasmin. These are
designated as intrinsic activation. Extrinsic activation occurs when the
endothelial cells of the vascular system release tissue activator, when
stimulated by certain activated clotting factors, exercise, hypotensive shock,
pharmacologic stimulators, and venous stasis.
65 DISCUSS THE ACTIVATION OF PLASMINOGEN TO PLASMIN.
Plasminogen activator factor (tPA) has a strong affinity for fibrin and it is
absorbed onto the fibrin polymers, then incorporated into the fibrin clot. The
tPA transfers from the fibrin polymers to the plasminogen molecule, activating
it. The active plasmin degrades the fibrin polymer by forming degraded fibrin
fragments. These fibrin fragments are capable of activating plasminogen and
the fibrinolysis process will continue. If plasmin is free in blood circulation, it
will proteolytically degrade several of the coagulation protein factors (V, VIII,
and XIII), the kinin system, and the complement system. If the plasminogen
activator is in the blood, it is called an intrinsic activator. Fletcher factor
(Kallikrein) is considered to be an intrinsic plasminogen activator. If the
plasminogen activator is from the epithelium of the vascular system, it is
known as an extrinsic activator or tissue activator. Activated factor X,
thrombin, platelet activating factor, bradykinin, and protein C can stimulate
the release of tissue activator. Tissue activator can also be found in the heart,
kidney, and other body organs.
66
BRIEFLY DESCRIBE HOW PLASMIN DEGRADATION OF FIBRINOGEN
DIFFERS FROM FIBRIN.
Plasmin does not distinguish between fibrinogen or fibrin. The degradation
process is essentially the same as outlined in Objective #63.
67
DESCRIBE HOW FIBRINOLYSIS IS CONTROLLED.
Fibrinolysis must proceed at a rate that does not interfere with the healing of
the wound and the dissolving of the clot. The molecules that act to inhibit the
process of fibrinolysis are as follows:
[1] 2 - Antiplasmin is a single chain 2 - glycoprotein synthesized in the liver. It
is the principle inhibitor of fibrinolysis. 2 - Antiplasmin forms a one-on-one
stoichiometric complex with either plasmin or plasminogen. When plasminogen
is inhibited, it cannot form plasmin. It is thought that plasmins enzyme sites
are blocked, preventing binding or dissolution of the plasmin substrate
molecules (fibrin, fibrinogen, factors V and VII, and thrombin). 2 - Antiplasmin
also inhibits plasma kallikrein and activated factors II, X, XI, and XII which are
serine proteases. If 2 - antiplasmin is deficient because of hereditary
condition, hemorrhage may occur due to lysis of fibrinogen and degradation of
factors V and VIII. If the problem is a pathology which involves excessive
clotting as with disseminated intravascular coagulation (DIC), the patients 2 antiplasmin may be depleted due to excessive conversion of plasminogen to
plasmin.
Stoichiometric describes a mass relationship of molecules to each
other. It is the relative proportion of reacting molecules that forms a
product. Two moles of hydrogen plus one mole of oxygen forms water
as its product. One 2 - Antiplasmin molecule plus one plasmin or one
plasminogen forms one product, the antiplasmin - plasmin complex.
[2] 2 - Macroglobulin is a plasma glycoprotein that combines slowly with
thrombin, plasmin, and kallikrein. It is considered to be an important backup

inhibitor when 2 -antiplasmin levels are low. This is a very large molecule and
it mode of action is to trap the plasmin molecule as it attempts to attach
proteolytically to a substrate molecule. The level of inhibition is not as
complete as for 2 - antiplasmin since the bound molecule can exert small
levels of activation. Deficiencies of this inhibitor are rare and medically these
patients are asymptomatic.
[3] PAI-1 is produced in the hepatocytes, endothelial cells, and
megakaryocytes. PAI-1 is found in the -granules of platelets. PAI-1 inhibits
tissue plasminogen activator (t-PA) and two-chain type glycoprotein (urokinaselike) plasminogen activator (tcu-PA) by neutralizing the molecule and
preventing it from forming plasmin.
The extrinsic plasminogen activators designated as urokinase-type
plasminogen activator exists as two types [1] scu-PA and [2] tcuPA. The scu-PA (single chain type glycoprotein (urokinase-like)
plasminogen activator) molecule does not activate plasminogen,
but tcu-PA does.
[4] PAI-2 weakly inhibits t-PA and tcu-PA, but not scu-PA.
[5] PAI-3 inhibits tcu-PA and activated protein C.
Note: PAI = plasminogen activator inhibitor.
68
DESCRIBE THE LEE-WHITE WHOLE BLOOD CLOTTING TIME.
This testing procedure is included for its historical interest. It will not be used
for test questions. This is a whole blood clotting time test that has been used
to monitor heparin therapy. Prepare three 8.0 mm diameter tests tubes for the
test. Four mLs of blood is drawn with a 20 gauge needle via a clean, nontraumatized venipuncture. One mL of blood is deposited in each of the three
test tubes and the last mL is discarded. The tubes are placed in the water bath
for three minutes. Begin looking for clot formation as follows: [1] Tilt tube #1
at 30 second intervals until the blood can be completely inverted without
spilling. [2] Repeat with tube #2, and [3] lastly with tube #3. Note that the
handling and tilting of blood hastens the clotting process. The results obtained
for tube #3 are reported. A prolonged clotting time is considered to be
indicative of a coagulation deficiency. The normal range for the Lee-White
clotting time is five to ten minutes. If the tube diameter is 9.0 mm, then the
normal range increases to six to thirteen minutes. This clotting procedure is
considered obsolete and is no longer performed. The prothrombin time (PT)
and activated partial thromboplastin time (APTT) tests involve the same
coagulation time principle and are more reliable. Comments . . . . Tube #1 may
be left in the water bath over a 2 to 4 hour period and observed for clot
retraction. The tilting of the tubes are to be gentle, always in the same
direction, and at the same angle.
69
DISCUSS THE ACTIVATED COAGULATION TIME (ACT) TEST.
The activated coagulation time (ACT) test is a modification of the whole blood
clotting time test. It is a test designed to be performed at the bedside of the
patient. It is used to monitor heparin therapy. The test requires the use of a
tube that contains an activator (example: diatomaceous earth) to which must
be added a minimum of 2.0 mLs of whole blood. The blood is mixed to
distribute the activator and timing begins as soon as the blood is collected. The
tube is tilted and observed until a clot is formed. The mean normal time for
the clot to form is 98 seconds. If a person is on heparin therapy for deep vein
thrombosis, the expected clotting time ranges from 180 seconds (3 minutes) to
240 seconds (4 minutes). A person on heparin therapy for cardiopulmonary
bypass operation should demonstrate a mean clotting time of 400 seconds (7
minutes).
70 BRIEFLY DESCRIBE THE ROLE OF UROKINASE AND STREPTOKINASE IN
HEMOSTASIS THERAPY.
Urokinase and streptokinase are thrombolytic drugs used to treat pulmonary
emboli and coronary thrombosis. These drugs will activate plasminogen to
plasmin. These drugs are usually given via IV infusion and can induce a high
degree of fibrinolysis.
71
DISCUSS THE BLEEDING TIME IN THE INVESTIGATION OF HEMOLYSIS.
The bleeding time is an in vitro testing procedure to detect platelet

dysfunction and von Willebrands disease which will prolong the bleeding time.
Because aspirin causes prolongation of the bleeding time in normal patients,
this procedure should not be attempted on patients who have taken aspirin
within the past eight days. Drug therapy may also prolong the bleeding time.
Thrombocytopenia prolongs the bleeding time.
The Duke method (introduced in 1910) is the least reproducible of the bleeding
time techniques. It is not longer performed in most laboratories. The Ivy
method replaced the Duke procedure in 1941, which employs a blood pressure
cuff on the arm of the patient. The cuff is inflated to 40 mm Hg. Two or
three lancet incisions (about 1 mm deep and 0.3 mm long) is made on the
inside forearm and the bleeding time of the two or three cuts averaged. This
method greatly improved the bleeding technique with more reproducible
results. Severed capillaries tend to collapse (vaso-spasm phenomenon) which
shortens the clotting time and is independent of platelet function. The Duke
method had three variables and the Ivy method eliminated the capillary
variable. This left the platelet function variable and the human variable of the
capillary puncture. In 1969, the template method was introduced in which a
mechanical device produces a standardized length and depth of cut (1 mm
deep and 3 mm long), removing the human variable. The template bleeding
time is reproducible and the preferred technique. Normal template bleeding
time values range from 2.3 minutes to 9.5 minutes. The direction of the cut on
the forearm should be consistent for all patients, either perpendicular
(vertical) or horizontal to the bend of the elbow. Horizontal incisions tend to
yield longer bleeding times. Caution: The template method can cause
mild to significant scarring in some patients.
72 EXPLAIN WHY A PHYSICIAN WOULD ORDER A THROMBIN TIME (TT)
TEST.
This test (synonym: thrombin clotting time) measures the availability of
functional fibrinogen. The prothrombin time will be prolonged when [1] the
fibrinogen level drops to 75 mg/dL to 100 mg/dL, [2] if heparin is present, [3]
if fibrin or fibrinogen degradation products are present, and [4] a thrombolytic
agent (as streptokinase) is present. The prothrombin time is not specific, but
can detect a Factor VII deficiency. If the physician suspects heparin inhibition,
then the thrombin time (TT) test will help to confirm this problem. The
procedure requires citrated plasma (one part 0.109 M sodium citrate in 9 parts
of whole blood). The specimen must be centrifuged to obtain platelet poor
plasma. The plasma must be kept cold and the test performed within four
hours after collection. Normal values are determined by the thrombin
concentration used. Reference ranges may be [1] 8 - 9 seconds, [2] 15 to 20
seconds, [3] less than 24 seconds, or [4] 20 to 25 seconds. Caution:
Thrombin is not a stable reagent, once it is made up and warmed to 37
oC (takes about three minutes), it must be used immediately (usually
within seven minutes).
73
DISCUSS THE CLOT RETRACTION TEST FOR HEMOSTASIS STUDIES.
This is a test that evaluates the ability of the clot to shrink, becoming denser,
and expressing serum from the clot. This phenomenon was associated with
platelet function. Normal clot retraction is dependent upon a normal number
of functional platelets, calcium, ATP, and minimum of 200.0 mg of
fibrinogen/L. Normally clot retraction is initiated 30 seconds after the blood
has clotted (does not flow) in the tube. At 60 minutes, there should be
obvious/visible retraction. At four hours, most of the retraction that will take
place has occurred. The clot retraction tube is held for 24 hours, at which
time, the retraction is considered to be complete. In the retraction process,
the clot will pull away from the sides of the tube and some free erythrocytes
may be observed at the bottom of the tube. In a normal retraction, the clot
should make up 45% to 50% of the total blood volume in the tube. If there is an
abnormality in the clot retraction process, the clot will occupy more than 50%
of the total volume in the tube. There may be no evidence of a discernable clot
or its retraction. One feature of abnormal clot retraction is the increased
fallout of RBCs. See illustrations of normal and abnormal clot retraction. Clot
retraction is abnormal when para-proteins are present (as in multiple

myeloma), thrombocytopenia, Glanzmanns thrombasthenia, Bernard-Soulier
disease, dysfibrinogenemia, and disseminated intravascular coagulation (DIC).
Observe the following illustrations of a normal and abnormal clot retraction.
This test is seldom performed in the laboratory. It is included to describe how
blood behaves in the clotting process.
Comment: The clot retraction test is generally considered to be of

little clinical use. The more sophisticated platelet function test have
made this procedure a seldom requested procedure.
74 EXPLAIN THE CONCEPT OF AGGREGOMETRY AS A PLATELET FUNCTION
TEST.
This testing procedure requires special aggregation reagents and a special
photometer with a stirring device. Blood must be collected with a plastic
syringe and stored in plastic tubes. The blood is centrifuged at as speed
equivalent to 150 X g for 30 minutes to prepare a platelet-rich plasma (PRP). If
RBCs appear to be present in the PRP, repeat this centrifugation step again for
5 minutes. A platelet poor plasma (PPP) must also be prepared by spinning at
1500 X g for 15 minutes. Cap the specimens in a plastic tube to prevent CO 2
loss and pH changes. Perform a platelet count to determine the platelet
concentration in the PRP. If the count is between 200 X 109/L and 300 X 109/L,
proceed with the aggregometry studies. If the count is greater than 300 X
109/L, adjust the sample by diluting it with PPP. Repeat the platelet count for
verification. A designated volume (usually 0.5 mL of PRP) is placed in the
aggregometer well or cuvette and appropriate aggregating reagent is added.
The optical density of the specimen is monitored and as aggregation takes
place the specimen becomes clear and the light transmittance increases. All
changes in optical density over time are recorded in a graph format as a curve.
If the curve deviates from the normal expected curve, the slope of the curve
changes and forms the basis for determining the amount or percent of platelet
aggregation. Examine the following illustration of the features of the platelet
aggregation curve.
The principle of platelet aggregometry is to deliberately add a platelet
aggregating agent to PRP, induce shape change and then cause the platelets to
aggregate or clump together. When the test is initially set up, the
aggregometer (type of photometer), the suspended platelets form a turbid
suspension. As aggregation takes place the test suspension clears and a change
in the light transmission is measured and recorded. The test procedure requires
that the patient be in a fasting state and that their last meal be fat free. The
patient should not be taking aspirin or non-steroid anti-inflammatory
medications. The patients blood is to be drawn with a plastic syringe and the
blood added to 3.8% sodium citrate. The blood to citrate ratio is nine parts
blood to one part anticoagulant. Testing for platelet aggregation should be
completed within two hours after collection. See the following illustration for
platelet aggregation scheme.
75
DISCUSS SIX PLATELET AGONISTS USED IN AGGREGOMETRY STUDIES.
EPINEPHRINE. Also called adrenalin, it produces a biphasic curve of

irreversible platelet aggregation. A small percentage of patients will
demonstrate a monophasic curve. Abnormal platelet curves are observed in
thrombosthenin, uremia, and storage pool disease.
Storage Pool Disease. This is a platelet disorder characterized by the
absence of dense bodies and/or -granules. Platelets cannot release ADP,
calcium, platelet factor-4, etc. This is observed in Wiskott-Aldrich syndrome,
thrombocytopenia (with absent radius bone in both arms) known as TAR, and
Chediak-Higashi syndrome. There is no treatment for this platelet disorder.
ARACHIDONIC ACID. When this reagent is added to the patients specimen, a
single monophasic curve is produced, resembling that produced by collagen. If
there is a platelet deficiency of thromboxane and/or cyclooxygenase, the curve
is suppressed. Aspirin inhibits this reagent resulting in the absence of a curve.
The patient must be aspirin free for eight days before testing. Abnormal curve
results are observed in thrombasthenia. Note: Arachidonic acid is heat and
light labile and must be stored in the frozen state away from light. It is
permissible to refreeze arachidonic acid aliquots three times.

COLLAGEN. When collagen is added to a patients specimen the reagent
initiates a monophasic curve after 30 to 60 seconds. This aggregation
phenomenon is dependent upon an intact platelet membrane and membrane
receptors, normal phospholipase pathway, and normal cyclooxygenase and
thromboxane functions. If an abnormal response occurs, this may suggest
disorders such as platelet membrane defect, storage pool disorders, aspirin-like
defects, thrombasthenia, and uremia. NOTE: Collagen should not be
frozen. Store at refrigerator temperature.
Aspirin-like defects is an anomaly that describes platelets
with normal appearing granules which do not have the
ability to release their contents. Deficiencies in
cyclooxygenase or thromboxane synthetase may be the
cause.
RISTOCETIN. Ristocetin is an antibiotic obtained from Nocardia lurida. The
action induced by this agonist produces a monophasic curve. A normal curve is
dependent upon intact platelet membranes, a functional von Willebrand factor
(vWF) receptor site, and ristocetin cofactor in the patients serum. Abnormal
curves may indicate von Willebrands disease (curves vary with the type of vWF
disease) or Bernard-Soulier syndrome.
THROMBIN. Thrombin reacts with several receptor sites on the platelet
membrane. A monophasic curve is produced in normal individuals. Abnormal
curves may indicate a storage pool defect disorder.
ADENOSINE DIPHOSPHATE (ADP). ADP produces a monophasic curve if used
in low concentrations, but a biphasic curve in high concentrations. Abnormal
curves are observed when aspirin is present, aspirin-like release defects,
afibrinogenemia, uremia, storage pool disease, and thrombasthenia.
76 ILLUSTRATE AND/OR RECOGNIZE AGGREGATION CURVES WITH
DIFFERENT AGGREGATING REAGENTS.
77
EXPLAIN THE PRINCIPLE OF THE PLATELET ADHESIVENESS TEST.
This is an in vitro procedure to evaluate the ability of platelets to adhere to

each other and to walls of damaged vessels. Two samples of blood is collected,
one specimen collected under standard procedures in an EDTA tube and the
other into a similar tube but requiring the blood to pass through a tubing
packed with glass beads. If the platelets are normal, the second tube will
contain from 40 to 75% of the platelets counted in the first tube. The adhesive
nature of the platelets will cause them to adhere to glass beads. The results
reported reflect the number of platelets that adhered to the glass beads.
Determine the difference between the two values and divide the difference by
the count without beads and multiply the result by 1000 to obtain the
percentage. Normal values reported are variable; from a low of 26% to a high
of 95% platelet adhesion. This test is difficult to interpret. Decreased values
are observed in [1] Glanzmanns thrombasthenia, [2] von Willebrand disease,
[3] Chediak-Higashi syndrome, [4] certain myeloproliferative disorders, [5]
uremia, and [6] ingestion of drugs and/or aspirin. Increased values are
observed in [1] venous thrombosis, [2] pulmonary embolism, [3] carcinoma,
[4] pregnancy, [5] splenectomy, and [6] patients taking oral contraceptives.
78
DISCUSS THE CIRCULATING ANTICOAGULANT TEST.
Some coagulation problems are due to the presence of inhibitors, something

that interferes with the coagulation mechanism, and are not due to a
deficiency of a coagulation component. These may develop because of some
disease state or an idiopathic problem. Such inhibitors are IgG antibodies (some
antibodies are reported of the IgM class). There are two groups of circulating
inhibitors:
[1] Specific inhibitors . . . . This group are antibodies and are directed
against certain coagulation factors. These often react in a progressive manner
and tend to be irreversible. The inhibitors that are seen the most are those
directed against factors VIII and IX.
[2] Non-specific inhibitors . . . . These are not directed against any specific
coagulation factor. Non-specific inhibitors may not be associated with bleeding.
These are immediate in action and will form a protein-protein complex with a
specific factor or group of factors. Such complexes are reversible and generally
do not destroy the factor(s). The lupus-like group of circulating inhibitors are
specific against phospholipids and do not cause bleeding. Another group of
inhibitors seen in this category are seen in patients with multiple myeloma or
benign paraprotein disorders. Liver disease may result in the development of
non-specific inhibitors.
These inhibitors can be detected by the activated partial thromboplastin time
(APTT) test and sometimes by the prothrombin time (PT) test. To determine if
the abnormal result is due to a coagulation factor deficiency or a circulating
anticoagulant or inhibitor, repeat the test by mixing one volume of the
patients citrated plasma with an equal volume of normal plasma. If the cause
is due to a coagulation factor deficiency, the APTT test result will be normal. If
circulating inhibitors are present the patients plasma will not be corrected and
the results will be prolonged. The reason that the patients plasma would be
corrected if the problem were a coagulation deficiency is that it requires about
50% of the coagulation factors to produce a normal test result.
79 EXPLAIN THE DIFFERENCE BETWEEN AGED AND ABSORBED PLASMA IN
COAGULATION STUDIES AND HOW TO INTERPRET THE RESULTS OBTAINED.
Aged serum is obtained by allowing a tube of clotted normal blood to incubate
at 37 0C for three hours. One procedure requires that to this tube of blood, add
one part of 0.109 M sodium citrate to nine parts of whole blood. Allow the
blood to incubate an additional two hours. Centrifuge for ten minutes and
remove the serum. The serum is ready to be used. The second procedure
requires that the clotted blood be centrifuged without the addition of
additives. If stored, store at -20 0C. When using aged serum, dilution may be
required. If so, use 0.85% NaCl. Aged serum contains factors VII, IX, X, XI, and
XII. Incubation destroys the labile factors.
Absorbed plasma is made by adding 100 mg of barium sulfate to each mL of
fresh oxalated plasma (use sodium oxalate). NOTE: Aluminum hydroxide gel
may be used but requires fresh citrated plasma, use sodium citrate. Mix the
mixture for ten minutes at room temperature, then place in the refrigerator
for an additional ten minutes. Centrifuge at 250 rpm for ten minutes and
remove the supernatant plasma. Barium sulfate or aluminum hydroxide will
absorb out factors II, VII, IX, and X. Absorbed plasma contains factors I, V, VIII,
XI, and XII.
To use either absorbed plasma or aged serum, mix equal parts with the
patients plasma and perform the APTT or PT test. Examine the following table
to see how substitution studies might reveal the factor deficiencies.
N = normal, A = abnormal, and * = testing not required.
Probable
ABSORBED PLASMA
AGED SERUM
factor
APTT
PT
APTT
PT
APTT
PT
deficiency
A
A
A
N
A
A
A
N
80
N
N
N
A
A
A
A
N
N
A
N
*
A
N
*
*
*
*
A
A
N
*
N
N
A
*
N
A
*
*
*
*
N
N
A
*
XI or XII
IX
VIII
VII
X
V
II
No deficiency
BRIEFLY DESCRIBE THE UREA SOLUBILITY TEST.
The urea solubility test is specific for factor XIII deficiency. It is also called the
Factor XIII Screening Test. Routine coagulation screening tests do not detect
factor XIII deficiency. To perform this test, add calcium to citrated plasma to
form a fibrin clot. Next add 5M urea to the clot and incubate at room
temperature for 24 hours. If there is no deficiency, the clot will remain intact.
If factor XIII is missing, the clot will dissolve in this period of time. This test
will detect a factor XIII deficiency if 98% of the factor is defective or missing.
81
EXPLAIN WHY A PHYSICIAN WOULD ORDER A STYPVEN TIME TEST.
Stypven is Russell viper venom that has thromboplastin-like activity. If this

reagent is added to plasma together with platelets and calcium chloride, the
coagulation process will begin at the factor X step followed by conversion of
prothrombin to thrombin, then fibrinogen to fibrin. This procedure will detect
deficiencies in factors I, II, V, and X.
82
EXPLAIN WHY A PHYSICIAN WOULD ORDER A REPTILASE TEST.
Reptilase is an enzyme from the venom of the Bothrops atrox viper and will
convert fibrinogen to fibrin. It is not affected by heparin. If a thrombin time is
prolonged because of heparin, this test can determine if fibrinogen is
functional/normal. The reptilase time and thrombin time will be prolonged in
hypofibrinogenemia, dysfibrinogenemia, streptokinase therapy, or when
fibrinogen degradation products are present.
83 EXPLAIN WHY A PHYSICIAN WOULD ORDER A FACTOR VIII:C INHIBITOR
ASSAY.
There are antibodies that can develop in patients with a factor VIII deficiency.
If they develop in such patients, they can provoke serious bleeding episodes.
They develop after sensitization by a factor VIII protein transfusion. Some
patients will develop high antibody titer (designated as high responders) with
repeated transfusions. This is an anamnestic response. Other patients are
almost non-responsive when other will demonstrate little or no response in
titer. These patients are designated as low responders. Factor VIII antibodies
can arise in patients without hemophilia for reasons not understood. Such
antibodies can arise in collagen vascular disease, dermatologic disease, drug
reactions, multiple myeloma, and Waldenstroms macroglobulinemia.
The presence of factor VIII antibodies in non-hemophilic patients can induce a
state of bleeding that mimics hemophilia. The clinical course of bleeding is
variable, from being very mild to a fatal episode. Bleeding is manifested by
dissecting hematomas, epistaxis, hematuria, post-traumatic hemorrhage, postsurgical hemorrhage, and hemarthrosis.
Laboratory findings usually begin with a prolonged APTT (measures all factors
except VII and XIII) and an normal PT (measures factors I, II, V, VII, and X). The
PT is prolonged in Factor V deficiency.
Treatment is dependent upon the titer of the antibody and it avidity for the
factor VIII antigen. Human, porcine, and bovine factor VIII concentrates and
vitamin K-dependent factors have been used as treatment strategies. There are
risks in using this approach.
84
EXPLAIN WHY A PHYSICIAN WOULD ORDER A EUGLOBULIN CLOT LYSIS
TIME.
He probably would not order this test. If he did, it would be to measure
fibrinolytic activity. Normal fibrinolysis occurs at a slow rate in the euglobulin
system. If a firm clot is present after 90 minutes, the fibrinolysis process is
normal. If the clot is lysing and deteriorating in the first 90 minutes, then
fibrinolysis is increased and abnormal.
The procedure is simple. Plasma is acidified with cold dilute acetic acid until
the solution attains a pH of 5.35 to 5.40. The next step is to refrigerate the
mixture for 30 minutes, in which time a precipitate should form that contains
factor I, plasminogen, plasmin, and plasminogen activators. This precipitate is
the euglobulin fraction. Centrifuge the tubes and decant the supernatant.
Resuspend the residue in borate buffer, then add thrombin. Begin timing.
Incubate at 37 oC. The clot will form in a short time. Check the clot every ten
minutes for lysis. If no lysis after 90 minutes, the test is complete. Report as
normal. If the time is shorter, then this indicates increased plasminogen
activator activity.
85 EXPLAIN WHY A PHYSICIAN WOULD REQUEST A TEST TO
DEMONSTRATE THE PRESENCE OR ABSENCE OF FIBRINOGEN DEGRADATION
PRODUCTS.
Fibrinogen degradation products (FDP), also called Fibrin Split Products (FSP)
are observed in patients with acute and chronic disseminated intravascular
coagulation, alcoholic cirrhosis of the liver, surgical complications, primary
fibrinolysis, late pregnancy, deep vein thrombosis, and pulmonary embolism.
The very presence of FSP in these examples indicate that a plasmin release has
occurred or is occurring. Pathologic degradation of fibrinogen and fibrin are the
result of increased plasmin activity and sets the state for abnormal secondary
bleeding. FDPs are significant because that they have hemostatic effects
(includes antithrombin activity and interference with platelet activity and
fibrin monomer polymerization) and can be life threatening. Fibrinogen
fragments into X, Y, D, and E fragments and other low-molecular weight
products. It is the X and Y fragments that exert the anticoagulant effects by
polymerizing with fibrinogen. The Y and D fragments obstruct the
polymerization of fibrin which results in soluble fibrin formation. The E
fragments is inhibitory to thrombin. In addition, all the fragments are capable
of adhering to platelet surface membranes and causing platelet dysfunction
with poor aggregation properties. These complexes can be detected by the
protamine sulfate test, ethanol gel test, and latex FDP assay.
86
DIFFERENTIATE BETWEEN PRIMARY AND SECONDARY FIBRINOLYSIS.
Primary fibrinolysis (PF), a rare event, is the degradation of fibrinogen. This

is a pathological state characterized by increased amounts of plasminogen
activators, increasing the amount of plasmin in circulation. This results also in

the splitting apart of factors V and VIII. The fibrinogen degradation products
form fibrin like threads slowly and require about 24 hours for the evidence of
clotting to appear. This is not a common occurrence but appears when
damaged cells appear or malignant cells (as in prostate cancer) are present,
surgery, shock, acute leukemia, or cirrhosis of the liver.
Secondary fibrinolysis (SF) occurs in conditions like disseminated
intravascular coagulation (DIC) where there is deposition of fibrin. Secondary
fibrin degradation products tend to polymerize earlier, requiring about 30
minutes to become observable.
There are four tests that will differentiate between these two types of
fibrinolysis.
[1 ] The Euglobulin Clot Lysis Time. It is significantly decreased in primary
fibrinolysis.
[2] Platelet count. It is increased in PF (>100 X 109/L). In SF the platelet
count is usually less.
[3] Antithrombin III assay. Levels will be normal in PF, but decreased in SF.
[4] D-dimer test will be negative in PF, but positive in SF.
87 EXPLAIN THE PRINCIPLE BEHIND THE PROTAMINE SULFATE DILUTION
(PSD) TEST.
Fibrin degradation products (FDP) are soluble entities that interfere with the
normal coagulation process. If protamine sulfate is added to plasma containing
FDPs, then a variation of a phenomenon known as paracoagulation takes place.
This means that the various FDPs will begin to dissociate from fibrin and other
sites and will polymerize in the presence of protamine sulfate to form a
gelatinous button. The button is comprised of X and YY fragments. Firbrin
monomers will also join this odd polymerization phenomenon. Note if FDP are
present in large quantities, the test will not work well. The protamine sulfate
(also true for ethanol) will displace the small FDPs resulting in spontaneous
polymerization. For this reason the protamine sulfate dilution test and the
ethanol gelatin test can be used to tests for FDPs. The better testing
technique is Agarose gel chromatography. Caution: the gel is sometimes
difficult to observe. Laboratorians who read the PSD test require
experience. False positives occur in females before and after menses and in
patients with advanced cirrhosis. This test does not detect just fibrinogen
degradation products. If fibrinogen is present in large quantities, it tends to
precipitate as an amorphous mass which occurs as large FDP complexes with
fibrinogen. This is not a gelatinous clot.
By definition, paracoagulation occurs when thrombin splits
fibrinogen into fibrin monomers. The fibrin monomers will
start form a fibrin mesh (development of the clot). The

fibrin monomers will bind to fibrinogen and or fibrin split
products to form a precipitate. The end product is not a
typical clot but an amorphous, gelatinous-like mass.
88 EXPLAIN THE PRINCIPLE BEHIND THE ETHANOL GELATION (GEL) TEST.
Fibrin monomers can be precipitated in plasma by the addition of 0.15 mLs (3
drops) of 50% ethanol to 12 drops of platelet poor plasma (PPP) that has been
pretreated with one drop of 0.1 N NaOH (to raise the pH to 7.7 or higher). The
ethanol is layered over the plasma (do not mix) and allowed to sit at room
temperature. Look for a line of precipitation or gel at the interface. If either of
these are present, it is a positive test for the presence of soluble fibrin
monomers. This is an indicator of a disseminated intravascular clotting (DIC)
problem, pulmonary emboli, deep vein thrombosis, acute myocardial
infarction, eclampsia, postpartum hemorrhage, malignant tumors, preeclampsia, renal failure, liver cirrhosis, surgical complications, etc. If you
observe an amorphous (without form) flocculation, the test is negative. If
nothing happens after one minute, the test should be allowed to sit for an
additional nine minutes. If strands or gel appears after ten minutes, add one
drop of 0.1 N NaOH. If a precipitate appears and disappears, ignore it . . . . this
particular phenomenon is of questionable significance and should be considered
as a negative result. If the precipitate or gel persists, then the test is positive.
The presence of the precipitate or gel is called paracoagulation. The procedure
is considered to be more sensitive that the protamine sulfate dilution test and
it is equally difficult to interpret. COMMENT: [1] If the fibrinogen level is
>400 mg/dL, the test will be a false positive. [2] Do not use oxalate
anticoagulants as they adversely affect the pH. [3] The presence of
heparin and/or erythrocytes will not affect the test results.
89
DESCRIBE THE PRINCIPLE OF OPERATION OF THE FIBROMETER.
It is a modified manual technique that uses electromechanical means of

detecting the clot. It uses two wire electrodes on a probe arm, one of which
has a back-and-forth (0.5 second) sweeping action through the reaction
mixture. When the fibrin clot forms, it is engaged by the electrodes, and
electrical contact occurs and the timer is stopped. Timing is initiated
automatically via its pipet or a manual switch. Its precision is 0.5 seconds. It
is no longer a primary instrument in the laboratory, giving way to the optical
detection instruments. Its role in the laboratory is that of a back-up
instrument. It does have one advantage over the optical instruments, it can
detect fibrinogen levels <50 mg/dL
90 LIST FIVE SOURCES OF ERROR THAT OCCUR WITH THE
ELECTROMECHANICAL METHOD OF CLOT DETECTION.
[1] Possibility of transferring activated coagulation factors on the probes

electrode into the next patients sample and shortening of the clotting time.
[2] Use of incorrect reaction wells or cups.
[3] Technical error on the part of the laboratorian.
[4] Incorrect collection of the specimen.
[5] Expired reagents.
91 DISCUSS THE OPERATION PRINCIPLE OF THE PHOTO-OPTICAL
COAGULATION INSTRUMENT, SUCH AS THE COAG-A-MATE, MDA, OR MLA.
These instruments come in a variety of models and use a photocell to detect
the fibrin clot. They have an advantage over the electromechanical detection
systems in that the photo-optical detector can detect a rapid and significant
increase in light transmission. Accuracy is expected to be 0.1 seconds. The
specimen and reagents are pipetted into the reactions wells in such a way that
maximum mixing is obtained. There is no moving probe to mix the system as
required in the fibrometer. Accuracy of reagent and specimen delivery is
maintained by sensor systems that cause the probe(s) to make minimal contact
with the fluids. The technology and sensitivity of these instruments permits
detecting clots in chylous and icteric patient specimens.
In detecting the forming clot, at the onset of the test, the light is transmitted
through the reaction mixture with little scattering. As the reaction proceeds
and more and more fibrin is incorporated into polymers, then into the clot, the
amount of light scattering increases proportionally. This creates an electrical
signal that is analyzed by algorithms. At some point in the reaction, the signal
becomes such that the instrument determines that the clot has formed. A timer
is linked into the reaction which is turned on when the plasma is added to the
reagents. A detection system also turns on at the same time and it will turn off
the timer when the formation of the clot occurs.
An algorithm is a series of algebraic equations. This
means that it is an ordered sequence of steps. Each step
must be completed before the next step can proceed. This
mathematical strategy is programmed into a computer as
a series of mathematical progressions to enable it to use
the degree of light scattering as a means to determine the
completion of the clot.
92 LIST SEVEN SOURCES OF ERROR THAT MAY OCCUR IN THE PHOTOOPTICAL DETECTION COAGULATION SYSTEM.
[1] The possibility of a rippling optic effect that occurs when reagents and
specimen are added to the reaction chamber and causing the instrument to
report a shortened clotting time. This effect is eliminated by a guard

interval that delays the end-point detection that is built into the test
procedure. During this time the instruments sets up a base line for
transmitted light.
[2] Some very abnormal specimens (those that are very lipemic, icteric, or
hemolyzed) may cause erroneous clotting times.
[3] Patients with fibrinogen levels < 50 mg/dL.
[4] Patient specimens containing elevated heparin concentrations.
[5] Precipitation of cryoglobulins if present in large amounts in plasma that is
allowed to sit at room temperature.
[6] Incorrect collection of the specimen.
[7] Expired reagents.
[8] Laboratorian error.
93 LIST FIVE SOURCES OF ERROR TO AVOID IN COAGULATION REAGENT
USE.
[1] Leaving caps/lids off of reagent bottles allowing evaporation to occur and
changing the concentration of the reagent.
[2] Ignoring expiration dates.
[3] Changing reagent lots and not re-evaluating appropriate control and
reference ranges.
[4] Improper shipping, storage, and handling of reagents from the
manufacturer to the vendor to the customer.
[5] Failure to reconstitute reagents to manufacturers specifications.
[6] Improper storage temperatures in the laboratory.
94 LIST NINE STEPS IN ESTABLISHING A POPULATION REFERENCE RANGE
FOR COAGULATION TESTING.
The criterion for the establishment of a population reference range is the same
for hematology and chemistry testing. Remember that commercial controls are
not permitted in establishing this range. The purpose of establishing this
specific range is to make accurate and unbiased generalizations about the
sample taken from the patient. The nine steps are:
[1] Use a minimum of forty subjects. One hundred is ideal.
[2] Select healthy volunteers.
[3] Volunteers must represent the demographics of the region in age, gender,
race, etc.
[4] Specimens collected from the volunteers must be handled the same
identical way as that for a patients specimen.
[5] Specimens should be collected and tested the same day.
[6] Use the same reagents and instrumentation as used for patients.
[7] Perform a frequency distribution histogram to determine if the sample
population falls within the limits of the Gaussian curve.
[8] Investigate the reason for any results designated as outliers.
[9] Perform statistical calculations to establish standard deviations.

95. DISCUSS PROFICIENCY TESTING IN THE COAGULATION LABORATORY.
This is a quality control program for interlaboratory testing to evaluate the
coagulation results. By comparing the test results between the patient and the
controls, the laboratory can access their performance to that of other
laboratories. This increased the reliability of the test results reported to the
physician. When comparing results with other laboratories, use those
laboratories that use the same testing instruments and reagents used in your
laboratory. In coagulation testing, the prothrombin time (PT) test and the
activated partial thromboplastin time (APTT) test are dependent upon the
reagent system.
Reviewed and Revised.

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BASIC CONCEPTS OF HEMATOLOGY

EXPLAIN THE BASIC CONCEPTS / PURPOSES OF HEMOSTASIS.

DESCRIBE AND/OR ILLUSTRATE THE THROMBOCYTE.

The thrombocyte (platelet) is an active sol-gel structure capable of

04 DISCUSS THE PERIPHERAL ZONE AND ITS FUNCTIONAL ROLE IN THE

b. Glycoprotein Ib, a receptor molecule for vonWillebrands factor.

DISCUSS THE GEL-SOL ZONE AND ITS FUNCTION IN THE PLATELET.

This mid-zone structure forms the cytoskeleton of the platelet. It may be

DISCUSS THE ORGANELLE ZONE AND EXPLAIN ITS FUNCTION.

C. Lysosomes contain acid hydrolases.

DESCRIBE HOW PLATELET ACTIVATION IS INITIATED.

Activation can be either transient, reversible, or irreversible. Platelet

[6] Collagen is a major water insoluble protein of the white fibers of

DISCUSS PLATELET ADHESION.

Adhesion occurs when a collagen fiber is exposed. Platelets attach to the

DISCUSS PLATELET ACTIVATION.

DISCUSS THE SECRETION PHASE OF PLATELET PLUG FORMATION.

DESCRIBE HOW ASPIRIN AFFECTS PLATELET ACTIVITY.

[1] It inhibits the production of platelet cyclo-oxygenase, an enzyme that is

[4] If a patient is taking aspirin as a therapeutic preventative measure, they

BRIEFLY DESCRIBE HOW CLOT RETRACTION OCCURS.

DISCUSS HOW PLATELET ACTIVATION MIGHT BE REVERSED.

22 EXPLAIN THE MEANING OF THE TERMS, PRIMARY HEMOSTASIS AND

activation of the intrinsic coagulation pathway. They require a negatively

Labile describes the tendency to be unstable; unsteady, not fixed. It denotes:

25 LIST THE COAGULATION FACTORS DESCRIBED AS SERINE PROTEASES

Plasma Thromboplastin Antecedent (XI)

26 LIST THE KNOWN CLOTTING FACTORS AND DESCRIBE THE FOLLOWING

Ionized Calcium (IV)

type of protein: not a protein

Antihemophilic Factor (VIII)

H. Christmas Factor (IX)

vitamin K dependency: yes

Stuart-Prower Factor (X)

Plasma Thromboplastin Antecedent (XI)

Hageman Factor (XII)

Fibrin Stabilizing Factor (XIII)

type of protein: double chain 2 - globulin

NOTE THAT ONLY FACTORS V AND VIII ARE LABILE.

DISCUSS AND / OR ILLUSTRATE THE EXTRINSIC COAGULATION PATHWAY.

The extrinsic coagulation pathway is an alternate pathway describing how

DISCUSS AND / OR ILLUSTRATE THE INTRINSIC COAGULATION PATHWAY.

This is a sequence of initiating coagulation events that occurs within the

DISCUSS AND / OR ILLUSTRATE THE COMMON COAGULATION PATHWAY.

EXPLAIN THE TENASE COMPLEX IN THE COAGULATION PATHWAY.

ILLUSTRATE HOW THE VARIOUS COAGULATION PATHWAYS INTERACT.

DISCUSS THE COMPLEX NATURE OF COAGULATION FACTOR VIII.

Factor VIII is a complex of several components and constitutes the largest

been recommended that this acronym be discontinued or interpreted with

A. 100 - 58 = 42 and 595 - 58 = 537 therefore 42 divided by 537 = 0.0782

DISCUSS THE IMPORTANCE OF HEATING IN COAGULATION TESTING.

DESCRIBE IN SIMPLE TERMS THE PROTHROMBIN TIME PROCEDURE.

BRIEFLY DESCRIBE HOW TO EVALUATE A PROTHROMBIN TIME TEST.

DESCRIBE COMMERCIAL THROMBOPLASTIN.

Also known as tissue factor III, thromboplastin is an organic extract of rabbit

DISCUSS THE PROTHROMBIN TIME (PT) AS A SCREENING PROCEDURE.

BRIEFLY DISCUSS THE PRINCIPLE OF COUMADIN THERAPY.

Coumadin is a warfarin compound and competes with vitamin K in the liver

When a physician prescribes coumadin therapy, a review of what medications