Renee Wilkins, Ph.D., MLS(ASCP)CM
MLS 322 Clinical Hematology
School of Health Related Professions
University of Mississippi Medical Center
What is hemostasis?
The process in circulation where the
blood is maintained within the blood
vessels
 Depends on the balance of

 Vascular
 Plasma coagulation factors
 Platelets
 Fibrinolytic system
Primary
Hemostasis
PLATELETS
Hemostasis

Primary hemostasis
 Blood vessel constricts (vasoconstriction)
 Platelets adhere and aggregate to damaged area
of the vessel wall
 Results in the formation of the platelet plug
 Platelets must be adequate in number and must
be functioning normally in order for the platelet
plug to form
○ Thrombocytopenia
○ Aspirin therapy
Platelets (thrombocytes)






Cellular fragments of
megakaryocytes that contain
granules
Circulate 9-12 days
2-4 μm
Normal platelet count is 150-400,000/μL
100,000 are needed to perform platelet function
testing
Thrombocytopenia is the most common cause of
abnormal hemostasis
Platelet

Alpha granules within the organelle
contain platelet derived growth factor,
platelet factor 4, FV, FXIII, fibrinogen,
vWF
Formation of platelet plug

Adhesion (1-2 seconds)
 Platelets adhere to collagen fibers and vWF to form a bridge
(glycoprotein Ib)

Activation
 Morphological and functional changes
 Increases internal Ca++, swelling occurs
 Glycoprotein IIb/IIIa is activated

Aggregation (10-20 seconds)
 Vasoactive substances like ADP and thrombozane A2 are released




along with platelet procoagulants
gp IIb/IIIa is exposed and binds to fibrinogen
Other platelets are stimulated to adhere and aggregate
When activation and primary adhesion have been achieved, platelets
commence discharge of granule contents. Granule contents include
ADP, adenosine triphosphate (ATP), serotonin, calcium, vWF, Factor V,
and fibrinogen
Once the plug is formed (1-3 minutes), the platelet mass consolidates to
a dense thrombus (3-5 minutes), then retracts and stabilizes (5-10 min)
fibrinogen
Platelet
glycoprotein Ib
von Willebrand factor
blood vessel wall/subendothelium
collagen
Primary platelet plug
platelets
fibrinogen
Platelets in coagulation
Platelet factor 3 (PF3) is a phospholipid
that resides on or within the platelet
 PF3 is required in the activation of
certain coagulation factors (to help form
thrombin)

Diseases of Primary Hemostasis
Bleeds from skin or mucous membranes
 Factor abnormalities are usually internal
 Disorders of the vascular system

 Abnormalities or damage of lining of blood vessels
or subendothelial structures
 Superficial bleeding (bruises, petechiae)
 Platelet count and factors are normal

Platelet disorders
 Thrombocytopenia (<50x109/L)
○ Asymptomatic to severe
 Thrombocytosis (>450x109/L)
Diseases of Primary Hemostasis

Inherited (functional disorders)
 von Willebrand Disease (vWD) [adhesion]
 Bernard-Soulier syndrome (adhesion)
 Glanzmann Thrombasthenia (aggregation)
 May Hegglin anomaly (quantitative)
 Disorders of pregnancy (quantitative)

Aquired
 Idiopathic thrombocytopenic purpura (ITP)
 Drug-induced platelet dysfunction
○ Aspirin induced platelet dysfunction
○ Aspirin resistance
von Willebrand Disease
 Inherited, up to 3% of U.S. population
 Qualitative or quantitative abnormality of von Willebrand
Factor (vWF) – fails to bridge gap (NOT a platelet disorder)
 vWF is a cofactor to Factor VIII (VIII:vWF complex)
 Excessively heavy or prolonged menstrual bleeding occurs
in 20% of women
○ vWD may be the cause of menorrhagia in as many as 20% of these
women
 Other symptoms include easy bruising and gingival
bleeding
 3 types with Type 2 containing 4 subtypes
Nature of vWF
The vWF molecule (protein) is a chain of
identical subunits called multimers
 vWF is synthesized in alpha granules of
megakaryocytes

 Primary hemostasis: vWF bridges GPIb/IX on
platelets and collagen to form the plug
 Secondary hemostasis: vWF binds to FVIII which
ultimately leads to fibrin formation

vWF in plasma varies in size, but most binds
with VIII in plasma (1:1)
 FVIII is formed in liver before release
vWD
glycoprotein Ib
Normal
Platelet
No von Willebrand factor
means no adhesion…
von Willebrand disease
blood vessel wall
On the flip side….
Giant
Platelet
No glycoprotein Ib
means no adhesion….
Bernard-Soulier
von Willebrand factor
blood vessel wall
Bernard-Soulier syndrome
A.k.a. giant platelet syndrome
 Rare autosomal recessive disorder
 <1 in 1,000,000
 Moderate to severe thrombocytopenia
 Abnormal platelet function due to lack of
GPIb/IX complex on platelet
 Homozygous individuals have lifelong
bleeding tendency; heterozygous have
not significant bleeding

Glanzmann Thrombasthenia
Rare deficiency of glycoprotein IIb/IIIa on
platelet
 gp IIb/IIIa binds fibrinogen and is essential
to platelet aggregation
 Platelet count normal, but bleeding
prolonged
 Fails to aggregate with agonists

 ADP
 Epinephrine
 Collagen
May-Hegglin anomaly
Moderate
macrothrombocytopenia
(large platelets, low #)
 Dohle-like inclusions in
WBCs
 Associated with
abnormalities of MYH9
gene

Disorders of Pregnancy

Incidental thrombocytopenia of pregnancy
 Occurs in 5-7% of pregnancies
 Poses no risk for neonatal thrombocytopenia

Thrombocytopenia from pre-eclampsia
 Pre-eclampsia is a hypertensive disorder of
pregnancy (7-10%)
 10-20% of pre-eclamptic women will develop
thrombocytopenia
 Prolonged bleeding
 Pathogenesis unknown
Idiopathic Thrombocytopenic
Purpura (ITP)--autoimmune






Children
No gender preference
Platelet count <20x109
Abrupt onset of bleeding
Infection 1-3 wks prior
Lasts 2-6 weeks
ACUTE






Adults
Females 3:1
Platelet count 30-80x109
Insidious onset of bleeding
No history of infection
Lasts months to years
CHRONIC
Drug induced platelet dysfunction



Aspirin inhibits prostaglandin production and
Thromboxane A2 (TXA2)
Aspirin irreversibly acetylates the cyclooxygenase
enzymes which prevents formation and release of
thromboxane A2
Platelets affected by aspirin continue to circulate
but are no longer capable of functioning
COX-1
Thromboxane
Aspirin resistance
Evidence suggests that significant
insensitivity (5% - 60%) to aspirin occurs
among patients with defined coronary
disease and stroke
 Some individuals may have to take
alternative drugs (i.e. Plavix®)

 Plavix (clopidigrel) affects the ADP receptor
(P2Y12) which inhibits gp IIb/IIIa receptor
that binds fibrinogen
Aspirin & Clopidogrel Effects
QUESTION

Cells involved in hemostasis are:
 Erythrocytes
 Granulocytes
 Lymphocytes
 Thrombocytes
QUESTION

Normal platelets have a circulating lifespan of approximately:
 5 days
 10 days
 20 days
 30 days
QUESTION

Aspirin affects platelet function by
interfering with platelets’ metabolism of:
 Prostaglandins
 Lipids
 Carbohydrates
 Nucleic acids
QUESTION

Platelet activity is affected by:
 Calcium
 Aspirin
 Hyperglycemia
 Hypoglycemia
QUESTION

Thrombocytopenia is a characteristic of:
 Classic von Willebrand disease
 Hemophilia A
 Glanzmann thrombasthenia
 May-Hegglin anomaly
QUESTION

Which of the following is the most
common cause of an abnormality in
hemostasis?
 Decreased plasma fibrinogen level
 Decreased Factor VIII level
 Decreased Factor IX level
 Quantitative abnormality of platelets
QUESTION

Which of the following is a true
statement about acute idiopathic
thrombocytopenic purpura (ITP)?
 It is found primarily in adults
 Spontaneous remission usually occurs
within several weeks
 Women are more commonly affected
 Peripheral destruction of platelets is
decreased
QUESTION

Which of the following is characteristic of platelet
disorders?
 Deep muscle hemorrhage
 Retroperitoneal hemorrhage
 Mucous membrane hemorrhage
 Severely prolonged clotting times
QUESTION

Thrombocytosis would be indicated by a
platelet count of:
 100 x 103/μL (100 x 109/L)
 200 x 103/μL (200 x 109/L)
 300 x 103/μL (300 x 109/L)
 600 x 103/μL (600 x 109/L)
QUESTION

vWF antigen can be found in which of
the following?
 Myeloblast
 Monoblast
 Lymphoblast
 Megakaryoblast
QUESTION

Alpha granules are found on the platelet
in:
 Peripheral zone
 Sol gel zone
 Organelle zone
 Membranes
Secondary
Hemostasis
COAGULATION FACTOR INVOLVEMENT
Secondary Hemostasis
Results in the formation of a blood clot
because of coagulation factors
 Forms fibrin network and thrombus that
stabilize the plug/clot
 Stops bleeding completely
 Lysis (fibrinolysis) of the clot begins and
final repair of injury takes place

Thrombin acts on fibrinogen to
form fibrin strands (clot)
Secondary Hemostasis
Coagulation factors are proteins (with
exception to thromboplastin and calcium)
 All are involved in generating insoluble
fibrin (cascade)
 They can be divided into 3 families based
on properties:

 Fibrinogen group (thrombin sensitive) –
fibrinogen, FV, FVIII, FXIII
 Prothrombin group (vitamin K dependent) –
Factors II, VII, IX, X, Protein C and Protein S
 Contact family – Factor XII, Factor XI,
prekallikrein and HMWK
Vitamin K
Vitamin K is inhibited
by warfarin
(Coumadin®)
 All factors in the
prothrombin group
are affected
 Individuals on
warfarin need to
monitor vitamin K
intake (hypercoag)

Factors in RED are affected by
vitamin K
QUESTION

Coagulation factors affected by
coumarin (warfarin) drugs are:
 VIII, IX and X
 I, II, V and VII
 II, VII, IX and X
 II, V and VII
ANSWER

Coagulation factors affected by
coumarin drugs are:
 VIII, IX and X
 I, II, V and VII
 II, VII, IX and X (vitamin K dependent)
 II, V and VII
Mechanism of Coagulation
1.
2.
3.
Generation of Thromboplastic activity
Generation of thrombin
Conversion of fibrinogen to fibrin
Pathways for the Coagulation Cascade
Intrinsic pathway
 Extrinsic pathway
 Common pathway

Intrinsic vs. Extrinsic

Intrinsic
 All factors are contained in the blood

Extrinsic
 Activated by tissue thromboplastin (FIII)
 Thromboplastin is released from damaged cells
and tissues outside the circulating blood
Intrinsic Pathway





Circulating blood contains all
components that lead to activation
of Factor X
HMWK (Fitzgerald) and
prekallekrein (Fletcher) activate
FXII to FXIIa
Factor XII activates FXI, which in
turn, activates FXI (now FXIa) in the
presence of Ca++ ions
Factor IX forms a complex with
FVIII (with Ca and phospholipid on
platelets) which activates FX
Factor XI and XII are “contact
factors” b/c their activation is
initiated by contact with
subendothelial basement
membrane that is exposed at the
time of a tissue or blood vessel
injury
Intrinsic Pathway
Also these complex reactions take place
slowly, they account for the most coag
activities in the body
 The APTT test can monitor the intrinsic
pathway
 The APTT measures:

Factors XII, XI, X, IX, VIII, V, II, and fibrinogen
Common Pathway Factors
Extrinsic Pathway



Occurs when tissue
thromboplastin (not in
blood) enters vasculature
Factor VII is activated to
VIIa by Ca++ and tissue
thromboplastin (FIII)
Factor VIIa can now
activate FX (common)
Extrinsic pathway
vascular injury
X
tissue thromboplastin + VIIa
Ca++
VII
X
V
Xa
(VVa)
II (prothrombin)
IIa (thrombin)
I (fibrinogen)
fibrin
Extrinsic Pathway
The extrinsic pathway can also quickly provide
small amounts of thrombin (leads to fibrin
formation)
 Thrombin can also enhance the activity of Factor
V and VIII (intrinsic pathway)
 The PT test (prothrombin time) monitors the
extrinsic pathway
 The PT measures Factors VII, X, V, II, and I

Common
QUESTION

Which of the following factors is used
only in the extrinsic coagulation
pathway?
 II
V
 VII
 VIII
ANSWER

Which of the following factors is used
only in the extrinsic coagulation
pathway?
 II
V
 VII
 VIII
Common Pathway





Begins with FX
Activation of FX is the point
where both the intrinsic and
extrinsic pathway converge
Once Xa is formed, it’s
cofactor (FV), in the presence
of Ca++ and PF3 convert
prothrombin to thrombin
Thrombin acts to convert
fibrinogen to fibrin
Factor XII stabilizes the clot
Interpreting abnormal tests
Disorders of the coag cascade

Hemophilia A
 Factor VIII deficiency (VIII:C subunit)
 Have normal VIII:vWF molecule
 vWD is deficient of VIII:vWF

Hemophilia B
 factor IX deficiency
 Mild, moderate, or severe (need to test)

Hemophilia C
 Factor XI deficiency
 4th most common inherited bleeding disorder
Factor XII deficiency
Causes prolonged APTT
 Normal PT, PFA 100 and TT
 No signs of a bleeding disorder
 FXII may be more involved with
inflammation since FXII deficient
individuals have higher rates of
infection.

Disorders of the coag cascade

Vitamin K deficiency
 diet, malabsorption disorders, liver disease
pregnancy
 Can prolong factor assays that include II, VII, IX,
and X…as well as the PT and APTT
 Found in leafy green vegetables
○ Green tea (712 μg)
○ Avocado (634)
○ Turnip greens (408)
○ Brussels sprouts (317)
○ Broccoli (200)
Anti-coags that can affect the cascade
aPPT
Direct thrombin
inhibitors:
-Hirudin (leaches)
-Argatroban
Vitamin K antagonists:
-Coumadin® (warfarin)
Heparins:
-Heparin
-Lovenox® (enoxaparin),
LMWH
Factor Xa inhibitors:
-Arixtra®
PT
Heparin
Heparin and its low molecular weight
derivatives are effective at preventing deep
vein thromboses and pulmonary emboli in
patients at risk
 Heparin binds to the enzyme inhibitor ATIII
causing a conformational change that
results in its activation through an increase
in the flexibility of its reactive site loop
 The activated ATIII then inactivates
thrombin and other proteases involved in
blood clotting, most notably FXa

Fibrinolysis….almost done
Fibrinolysis is the last stage of coagulation
that causes the dissolution of the fibrin clot
 Fibrinolysis is mediated by the conversion
of plasminogen to plasmin
 Activation of plasminogen can be due to:

 Intrinsic activation- initiated by FXIIa & kallikrien
 Extrinsic activation- stimuli like vascular injury,
ischemia, exercise and stress
 Exogenous (therapeutic) activation- drugs like
streptokinase, urokinase, tissue plasminogen
activator (tPA)…for strokes
FIBRINOGEN
FIBRIN
Fibrin monomer
thrombin
D
E
Fibrin polymer
D
Factor XIII
plasminogen  plasmin
Crosslinked fibrin
polymer
plasmin
X fraction:
Y fraction
“Stabilized Clot”
Degradation products
D fragments
D-dimer (crosslinked D-domains)
D fragments
E fragments
Fibrinolysis testing
Most commercial FDP kits detect D & E
fragments which are both products of
fibrin and fibrinogen degradation (latex
kits)
 The D-Dimer test is a specific marker for
plasmin degradation of fibrin (latex kit or
ELFA)

Latex particles are coated with anti-human fibrinogen
SPR – Solid
Phase Reaction
Sample
goes here
Fibrinolysis

Disease states can either increase or
decrease fibrinolytic activity
 Disseminated Intravascular Coagulation
 Trauma from surgical procedures or
accidents
 Deficiencies in or consumption of the
various inhibitors and activators of the
fibrinolytic system
DIC
Uncontrolled formation of thrombi/fibrin
 Fibrinolysis is activated but rapidly
consumed thus leading to depletion of
coag factors and platelets
 Bleeding, shock, microscopic thrombi

 Petechiae, hematomas, deep tissue bleeding

Associated with obstetric emergencies,
septicemia, burns, crush injuries, cardiac
and vascular disorders
DIC lab findings
PT and PTT usually prolonged, not
always
 Fibrinogen – 2-3X normal (150-400)
 Platelets – thrombocytopenia (<50,000)
 FDPs – usually elevated
 D-Dimer – elevated

DVT
Occurs when a blood
clot forms in one of
the large veins,
usually in the lower
limbs
 The blood clot can
break loose and
move into the:

 Lungs (Pulmonary
Embolism)
 Brain (Stroke)
DVT facts





DVT occurs in about 2 million Americans every
year.
Up to 600,000 people are hospitalized in the U.S.
each year for DVT.
Fatal PE may be the most common preventable
cause of hospital death in the United States.
In the elderly, DVT is associated with a 21%
mortality rate, and PE is associated with a 39%
mortality rate.
PE is the leading cause of maternal death
associated with childbirth. A woman's risk of
developing emboli is six times greater when she is
pregnant
Lab tests

D-Dimer is most useful b/c it tests for the
breakdown of the fibrin mesh
 >10,000 critical (>500 pos)

FDPs are sometimes useful in post-op
deep vein thrombosis
Natural anticoagulants

In vivo anticoagulants help to prevent
thrombosis
 Antithrombin III (ATIII)
○ Inhibits thrombin and factor Xa, but can also affect
Factors IXa, XIa, and XIIa
○ Deficiencies can be inherited or acquired
○ ATIII can be given as treatment for thrombotic disorders
 Heparin Cofactor
○ ATIII heparin cofactor and HC-II are thrombin inhibitors
that are present in human plasma
○ They are produced by mast cells
○ Unfractioned heparin binds to ATIII to target factors IX,
X, V, and II (prothrombin)..detected by APTT or both (if
dose is high
Natural Anticoagulants (cont’d)

Protein C and Protein S
 Protein C is made in the liver and circulates
as zymogen
 It is converted to activated protein C (APC)
by thrombin and thrombomodulin
 APC with its cofactor, protein S, can
inactivate FV and VIII
Protein C/S Disorders

APC Resistance
 Also factor V Leiden
 Factor V Leiden is a genetic mutation that
alters Factor V so that APC cannot bind and
inactivate it
 This can lead to thrombophilia
(hypercoagulability…..thrombosis)
 20-50% of inherited thrombophilias
 Discovered at the Univ. of Leiden
(Netherlands)
QUESTION

A deficiency of protein C is associated
with which of the following?
 Prolonged APTT
 Decreased fibrinogen (<100)
 Increased risk of thrombosis
 Spontaneous hemorrhage
ANSWER

A deficiency of protein C is associated
with which of the following?
 Prolonged APTT
 Decreased fibrinogen (<100)
 Increased risk of thrombosis
 Spontaneous hemorrhage
QUESTION

Which of the following is a characteristic
of Factor XII deficiency?
 Negative bleeding history
 Normal clotting times
 Decreased risk of thrombosis
 Epistaxis
A coagulation lab performs:

Routine Testing








PT(INR)
aPTT
Fibrinogen
D-Dimer
FDP
Thrombin Time
PFA-100
Bleeding Time

Special Coag















Factor Assays
Lupus Panels
Mixing Studies
ATIII
Plasminogen
von Willebrand
LMWH
Euglobulin clot lysis
Factor XIII
Factor Inhibitor
Fletcher Factor
APC Resistance
Antiphospholipid testing (ELISA)
HIT (ELISA)
PAI-1 (ELISA)
Coagulation Basics

3.2% sodium citrate tubes

Anticoag to blood ratio is crucial
 a short draw could prolong results

Ratio is 9 parts whole blood to 1 part anticoagulant
(sodium citrate)

Discard first tube b/c it may be contaminated with
tissue fluid (tissue thromboplastin may activate
extrinsic system..prolonged PT)
Anticoagulants to remember
 Anticoagulants (EDTA, citrate) remove
calcium to prevent clotting (in vitro)
 Heparin (FIX, X, V, II) and warfarin (vitamin
K dependent factors II, VII, IX, X) prevent
the conversion of prothrombin (factor II) to
thrombin (in vivo)
Centrifugation

Samples are spun in a refrigerated centrifuge fro
10-15 minutes at 3500 rpms to obtain platelet poor
plasma
 Refrigeration maintains labile factors VIII and V

Samples for assays that are performed at a later
date are:
 Immediately separated
 Checked for platelet count (<10x109)
 Phospholipids from lysed platelets can falsely effect
phospholipid based tests (Lupus testing)
 frozen rapidly at -72°C
 Thawed rapidly to avoid cryoprecipitation (VIII)
Automated Testing




Automated (photooptical) or semi-automated
End point of reaction is a clot (measured in
seconds)
Clot formation is timed automatically and is
detected by a photocell that reads the optical
density change when the clot is formed
Contains a heat block to bring reagents and
plasma to 37°C
 Reagents not in use are kept at 4-6°C to maintain integrity
(on instrument, in fridge, or cooling block)


Analyzers automatically pipette (or require manual
pipetting)
PT, APTT, factor assays, fibrinogen, etc
STart 4 Analyzer
Electromechanical method
PT testing
Prothrombin time (extrinsic & common
pathways)
 Method of choice for monitoring Coumadin
(Vit K antagonist)
 Reagents include

 Calcium Chloride
Contained
together in a
 Thromboplastin
single vial
 Both of these are needed to initiate the extrinsic
pathway and test for any deficiencies
PT Testing
Normal PT = 10-14 seconds
 In the past, many labs performed a PT
test with (thromboplastin) and a
separate PT(INR) test for those on oral
anticoagulant therapy to determine an
INR
 Now, some labs run the PT(INR) on all
patients and have discontinued the
routine PT

PT INR
INR – International PT standardization for
oral anticoagulant therapy monitoring
 The WHO and the International Committee
on Thrombosis and Hemostasis
recommend that PT tests run on patients
on oral therapy use an INR value
 INR values are independent of the
reagents and methods used, and are
specifically intended for assessing patients
stabilized on long term oral anticoagulant
therapy

PT(INR)

To standardize the PT, a calculation is
used to obtain the International
Normalized Ratio (INR) for people on
warfarin (normal INR = up to about 4)
Patient PT
INR =
X ISI
Mean PT (of normal population)
International Sensitivity Index
Current normal PT in lab = 9.4-12.5
PT INR

Note:
 When coumadin is initiated, with a loading dose,
there is a rapid depletion of Factor VII during the
first 48 hrs, with other factors not being fully
affected until about 5 days after
 Prolonged PT times before 5 days have no
correlation with any therapeutic benefits
 Measurements are not suggested until the 5th
day of therapy
QUESTION

The prothrombin time (PT) test requires
that the patient’s citrated plasma be
combined with:
 Platelet lipids
 Thromboplastin
 Ca++ and platelet lipids
 Ca++ and thromboplastin
ANSWER

The prothrombin time (PT) test requires
that the patient’s citrated plasma be
combined with:
 Platelet lipids
 Thromboplastin
 Ca++ and platelet lipids
 Ca++ and thromboplastin
APTT Testing
Activated Partial Thromboplastin Time
 Screens for deficiencies involving factors of
the intrinsic pathway

 Most sensitive to Factors VIII, IX, X, XI & XII
 Can be prolonged in severe Factor V deficiency

Fresh citrated plasma provides all factors
necessary for the intrinsic clotting
mechanism except
 ionic Ca (removed by citrate)
 platelet factor (removed by centrifugation
APTT testing

An activator and phospholipid is incubated with
patient plasma to activate contact factors (creates
negatively charged surface)
 Activators: kaolin, celite, ellagic acid, silica
 Phospholipids: rabbit brain, cephalin, soy bean



Calcium is added after incubation and the time it
takes for a clot to form observed
Normal aPTT = varies from about 24-34 seconds
Actin® is a reagent that contains ellagic acid and
rabbit brain
APTT

Note:
 Heparin can be given continuously (IV) or in
subcutaneous mini-doses
 Care should be taken not to obtain samples
above heparin line
 Patients should be monitored prior to next
dose if APTT is to be meaningful
 Heparin contamination can sometimes
severely prolong APTT (>60 seconds)
Another APTT reagent: PSL

Panthromtin SL is more sensitive than
the routine APTT reagent
 It is used in some labs to detect inhibitors
affecting the APTT
 The “SL” refers to Lupus and is used to
detect risk of disease
 If prolonged and heparin is ruled out, further
Lupus testing is performed
Fibrinogen Testing





Thrombin converts fibrinogen to fibrin
Thrombin is added to plasma and the time
it takes to clot (determined in seconds) is
proportional to the fibrinogen concentration
in plasma
Calibration curves are needed for each
new lot of thrombin
Normal range = 150 – 400 mg/dL
Anything >500 is run manually on a semiautomated instrument (i.e. Start 4)
Factor Assays
Allows testing for specific factors by
making a series of dilutions on a control,
reference (deficient) plasma, and patient
 3 (automated) dilutions made: 1:10, 1:20,
1:40
 Run a PT or APTT, depending on what
factor is being tested
 The factor deficient plasma has every
factor except the desired factor

Factor assays
PT: 2, 7, 5, 10
PTT: 9, 8, 11, 12
The 3 points of the reference (calibration curve) is
plotted and a best fit line is drawn (automated)
 The 3 points of the patient or control are plotted
and compared to reference



 If patient is below reference, then the factor activity is
low (normal 50-150%)
 If patient is above, then factor is high (no worry)
 If patient crosses the reference line (not parallel), then
an inhibitor is suspected
Factor graph (basic example)
1:40
Inhibitor present,
possibly FVIII inhibitor
or Lupus anticoagulant
1:20
1:10
reference
Curvilinear paper is used to obtain straight line
Low factor
Factor XIII
Stabilizes clot
 Uses 2 tubes containing plasma are clotted
with Calcium chloride

 5M urea added to one tube
 Acetic acid added to the other

Checked (in water bath) at 30 min intervals
to monitor clot
 If clot dissolves in 10-30 min, FXIII deficiency
 Normal clots remain over an hour
 Umbilical stump hemorrhage is the hallmark of
FXIII deficiency
Lupus Anticoagulant Panel
LA (or inhibitor) is thought to be a
nonspecific phospholipid antibody
 Prolonged PTT is a routine screening
test
 If after testing, a lupus inhibitor is
detected, mixing studies are performed
to rule out factor deficiency

LA testing

Lupus panel consists of:
 APTT: Actin FSL and Pathromtin SL
 Dilute Russell’s Viper Venom Test (DRVVT) – modified
APTT that uses viper venom as an activator. Very sens.
 DRVV Confirm – higher concentrations of phospholipids.
This higher concentration of phospholipids allows the
reagent to “correct” the prolonged DRVVT.
 Anti-cardiolipin, anti-phosphatidylserine, anti-prothrombin
and beta-2-glycoprotein
 Sta-Clot kit detects lupus inhibitor
○ Tube A is an APTT
○ Tube B is an APTT with phospholipid
○ If clotting changes more than 8 seconds, it is positive
Triturus tests (ELISA)
Antiphospholipid tests (for APS
syndrome, SLE, etc)
 HIT Testing
 PAI-1 Inhibitor

HIT

Heparin Induced Thrombocytopenia
 Tests for anti-platelet antibodies to the PF4
receptor on platelets
 In patients on heparin or who have had
previous exposure to heparin
 Platelet count dramatically drops by 50%
after second exposure
 Must be removed from heparin and given
alternative treatment (i.e. argatroban,
LMWH)
Euglobulin Lysis
Overall screen of the fibrinolytic system
Fibrinogen, plasmin and fibrinolytic agents are
precipitated (left in tube)
 Inhibitors of fibrinolysis are discarded
 The precipitate is re-dissolved in buffer and
thrombin is added to form a clot
 It is put in a water bath and monitored every 10
min for 1 hour then at 2 and 24 hours


 Normal patient >2 <24 hours
 Abnormal <2 hrs increased fibrinolytic activity
 >24 hours decreased fibrinolytic activity
ATIII testing
Naturally occurring inhibitor that
neutralizes the serine protease
thrombin, FIXa, Xa, Xia and XIIa
 The inhibition of thrombin by
antithrombin is greatly accelerated by
heparin
 Chromogenic assays that inhibit
thrombin or Fxa, microlatex assays,
nephalometry are frequent assays.

Bleeding Time







Tests for platelet function
Normal 3-9 minutes
Patient MUST have a Plt count >50,000
Blood pressure cuff is applied (40)
Small incision made with lancet and the bleeding is
“wicked” with paper disc every 30 seconds until plug
forms
Test is terminated if bleeding does not stop after 15
minutes
Abnormal bleeding disorders include:




Thrombasthenia
vonWillebrand disease
Bernard Soulier
Aspirin sensitivity
PFA 100®



Platelet Function Analyzer
Simulates in vivo conditions
Uses 2 cartridges:
 Col/EPI
 Col/ADP

Could replace the bleeding
time


Francis J., Francis, D., Larson, L., Helms, E. & Garcia, M.
(1999). Can the Platelet Function Analyzer (PFA)-100 test
substitute for the template bleeding time in routine clinical
practice? Platlets, 10(2-3), 132-6.
Assesses platelet dysfunction
due to:
 ASA-like platelet defects
 congenital platelet defects
(vWD)
PFA 100
Platelet
aggregation/plug
800 µL
blood
Filter with
Epinephrine or ADP
Once started, a timer begins. When
platelets have plugged aperature, the final
time is reported as “closure time”
Interpretation
Col/EPI
True platelet
defect (vWD,
etc)
Aspirin-like
defect (ASA)
Col/ADP
+++
+++
(ex. >300)
+++
normal
Platelet Aggregation
A photo optical instrument or
instruments using electrical currents are
used to detect aggregation of platelets in
platelet rich plasma or whole blood,
respectively
 PRP or whole blood is mixed with an
aggregating agent and results are
transmitted to a chart recorder

 Collagen, ADP, epinephrine, thrombin,
serotonin, ristocetin
Platelet aggregation curves
http://www.practicalhaemostasis.com/Data%20Interpretation/Data%20Questions/data_interpret
ation_platelet_function.html click here for practice cases
Aggregation Disorders
Glanzmann’s
Thrombasthenia
Bernard-Soulier
Syndrome
ADP
Abnormal
Normal
Thrombin
Abnormal
Abnormal
Collagen
Abnormal
Normal
Epinephrine
Abnormal
Normal
Ristocetin
Normal
Abnormal
vWF
Normal
Abnormal
Lab Findings
Platelet Aggregation
QUESTION

Heparin Induced Thrombocytopenia
(HIT) is an immune mediated
complication associated with heparin
therapy. Antibodies are produced
against:
 ACLA
 PF4
 AT
 B2GP1
QUESTION

A bleeding time is used to evaluate the
activity of:
 Platelets
 Prothrombin
 Labile factor
 Factor XIII
QUESTION

A patient has been taking aspirin
regularly for arthritic pain. Which one of
the following tests is most likely to be
abnormal in this patient?
 Platelet count
 Template bleeding time
 Prothrombin time
 Activated partial thromboplastin time
QUESTION

Biological assays for antithrombin III
(ATIII) are based on the inhibition of:
 Factor VIII
 Heparin
 Serine proteases
 Anti-ATIII globulin