Fundamentals of
Coagulation Testing
Robert Gosselin, CLS
University of California, Davis Health System
Department of Clinical Pathology and Lab Medicine
Sacramento, CA
Robert.gosselin@ucdmc.ucdavis.edu
Goals and Objectives
• The more painful first one…
– Quick review of hemostasis
– Technical aspects of laboratory tests
• Methods
• Limitations
• Outside the lab stuff
• Subsequent presentations
– Disease states and laboratory tests for
Dx
• Review session, case studies, lab
test caveats…
Goals and Objectives
• Resident driven…
– What do you want?
– What do you need?
– Bringing questions to the table…
Hemostasis
• Cellular
– Endothelium
– Platelets
– Red blood cells
– White blood cells
• Fluidic
– Procoagulant factors and regulators
– Fibrinolytic factors and regulators
Role of endothelium
• Procoagulant
– Collagen
– vWF stored in WP bodies
– Tissue factor expression
– Cytokine/chemokine
Role of endothelium
• Anticoagulant
– Heparin sulfate
– In presence of thrombin
• Prostacylcin production  PLT aggregation
• Nitric oxide- vasdilation  PLT aggregation
• Express thrombomodulin  thrombin
production
• Release TFPI  thrombin production
• Release plasminogen activators 
fibrinolysis and promote wound healing
Cytokines/Chemokines
•
•
•
•
•
TNF-α
IL1-β
TF expression
IL-6
IL-8
CD54 - transmembrane adhesion molecule to
facilitate WBC to endothelium
• CD62E and CD62p - adhesion properties
• Complement
– C3a C5a induce IL release from endothelium
– C3a:C4aC5a induce elastase release
Cellular components
• Platelets
– Receptors
• Adhesion
• Aggregation
• Factors
– Intracellular
• Adhesion
• Procoagulant factors
AD Shapiro,
WFH 1999;19
Role of platelets
collagen exposure
P selectin exposure
Monocyte adhesion
vWF-GPIb-IX
GPIIb-IIIa
Fbg
B-TG
Fbg,
Platelet
Release of ATP,ADP
TF serotonin,
IX
Thrombin
Cellular components
• RBCs
– Facilitate platelets to the endothelial
surface
– Thromboplastin source
• WBCs
– Tissue factor expression-MC
– Cytokine expression
– Elastase production-PMNs
Revised Cascade
Tissue Factor
VII
VIIa-TF
X
VIIa-TF
IX
XIa
IXa
neutralized
VIIa-TF-Xa
VIIIa
Xa
VIII
TFPI
V
Va
Prothrombin
Thrombin
F1.2
Cytokine release
THROMBIN
Procoagulant
Fibrinogen cleavage: fibrin
monomer generation
Activates factor VIII and V
Anticoagulant
Complex with thrombomodulin:
protein C activation
Generate TAFI:
fibrinolysis inhibitor
Induces platelet aggregation
Endothelial release of tPA
Activate factor XIII
Thrombin
XIII
Fibrinogen
Fibrin
XIIIa
FDP
Stabilized
Fibrin
Plasminogen
uPA or tPA
Plasmin
uPAR
FDP
uPAR:uPA
CLOT LYSIS
MMP
Extracellular
degradation
D-dimer
D
E
FIBRIN
E
D
D
E
D
D
D
Plasminogen
tPA
D
D-dimer
Plasmin
uPA
FIBRINOGEN
D
D
E
D
Fragment X
E
D
Fragment Y
D
Fragment D
E
Fragments D & E
HMWK
Pre
Traditional “Waterfall” Cascade
XII
XIIa
XI
XIa
IX
IXa
VIII
Ca+2
VIIa
VII
TF
PF4
Ca+2
VIIIa
X
Xa
V
Va
PF4
Ca+2
Prothrombin
Thrombin
F1.2
Fibrinogen
Fibrin
FPA
FPB
HMWK
Pre 
XII
XIIa  
XI
Primary Coagulation Regulators
AT 
XIa  
PC 
VII
Ca+2
IX
VIII
IXa 
VIIIa  
VIIa 
C1 Inh 
PF4
Ca+2
HCFII 
TFPI 
Xa  
X
V
PS 
TF
Va  
Prothrombin
Fibrinogen
2-MG 
PF4
Ca+2
Thrombin   
Fibrin
Thrombin
XIII
Fibrin 
Fibrinogen
XIIIa
FDP
Stabilized
Fibrin
Plasminogen
PAI-I
TAFI
2-AP
2-MG
uPA or tPA 
uPAR
Plasmin

FDP
uPAR:uPA
CLOT LYSIS
MMP
Extracellular
degradation
D-dimer
Tests for endothelial function
• Nada…
– All indirect measurements
• Soluble factors
– vWF, Endothelial-1
– Cytokine
Test for WBC and RBC
function
• WBC
– Indirect assessment
• Soluble factors
• Flow cytometry
– Aggregates
– Cytokine production
• RBC
• HCT
Tests for platelet function
• In addition to absolute numbers
– Platelet function testing
• Screening methods
– PFA and others
• Aggregation methods
– Whole blood versus PRP
• Flow cytometry
– All kinds of possibilities…
» Drug occupancy
» Activation
Platelet Function Assessment
• Chronolog Corporation
– Single channel impedance method
• Dade Behring Incorporated
– Shear rate induced aggregation
• Medtronics
– Modified ACT using PAF
• Accumetrics
– Optical detection of fibrinogen coated beads
• Hemadyne
Platelet contractile force
• Helena
– Plateletworks-changes in impedance (2 tubes)
• Diamed
– Cone and Plate(let)
Principle of the PFA-100®
• Collagen/Epinephrine
— primary
Results reported(CEPI)
as “Closure
time”screening
in secondscartridge
• Collagen/ADP (CADP) — differentiates dysfunction due to aspirin
Coagulation methods-functional
• Clot detection
– Screening versus specific
• e.g. aPTT versus factor VIII
– Light scatter vs mechanical
• Chromogenic
• Immunologic
Coagulation methodsantigenic
• ELISA
• Electrophoresis
• Immunologic
Coagulation Testing in the Clinical
Laboratory
Prothrombin times (PT)
PP Plasma
+
Activator (+ CaCl2) Clot Detection
37oC
Activated partial thromboplastin times (aPTT)
PP Plasma
+ Activators
37oC
CaCl2
Clot Detection
Optical
methods:
change in
turbidity
Mechnical
methods:
change in
motion
optical density
Clot based tests
time
CRUMMY BLOOD DRAW
Increased activation
Delays in testing:
optical density
 PF4 release
 Factor levels
Factor VIII
Fibrinogen
Drugs
Fxs
DDAVP
PCCs
Novo7
Foods (e.g. caffeine)
Exercise
(Physiological) Stress
time
 Factor activity
Drugs- UFH, DTI, Xigris
Lipemia, icterus
Crummy blood draw:
optical density
Factor consumption
Delays in testing:
+/- Lupus
anticoagulant
Inhibitors
 Fx VIII activity
time
Anti-Xa activity
plasma [heparin] + exogenous antithrombin
Excess fXa
AT-heparin-Xa complex + residual fXa
Chromogenic substrate
yellow color
HIT antibodies IgG (+)
Conjugated Anti-human IgG antibody ¤
Chromogenic tag 
Wash
Microwell
containing
target
antigen:
PF4-heparin
complex
+
Wash
¤ ¤
¤
++ +
+ +
Incubate

 
¤
+ ¤
+ ¤
+
Incubate
Color
Amountofofcolor
Amount
color
proportional toto
proportional
amount of
amount
ofantibody
antibody
present
present
Reagent beads coated with
anti-vWF
Patient
vWF 


 



Testing well








Incubate
Instrument reading—
changes in optical density
secondary to aggregates
Test Validation
Accuracy
Precision
Reportable range (Linearity)
Verify manufacturer’s
reference interval (normal range)
Notes
Comparison of new method to existing method or
reference method.
Determining the precision of running a single sample
multiple times concurrently (within-run precision) as well
as the same sample (usually control material) over a
period of days (day-to-day precision) to determine the
coefficient of variation (CV).
Assesses the reportable range (high and low), as well a
reproducibility of diluted samples
Appropriate for laboratory’s patient population.
Pediatric reference ranges may be cited from
acceptable references
If modifications to an FDA-approved test is used, or if
in-house test is used, then additional performance
characteristics must be evaluated and documented to
include aforementioned accuracy, precision, reportable
range, reference intervals, but additionally:
Analytical sensitivity

Analytical specificity, including interfering

substances
Other performance characteristics required

for testing
XII
HMWK
XIIa
Pre
XII
XI
XIa
VII
XI
Ca+2
TF
IX
VIII
IXa
VIII
VIIa
PF4
Ca+2
VIIIa
X
X
Xa
V
VII
IX
Va
Prothrombin
V
PF4
Ca+2
II
Thrombin
Fibrinogen
aPTT
Fibrin
PT
aPTT
• Screen for factor deficiencies
• Monitoring drug effect:
– UFH
– DTI
– Factor VIII & IX replacement Rx
• Other
– LA
PT/INR
• Factor deficiency
• Drug monitoring
– Oral vitamin K antagonist
• Effect of UFH Rx varies
– Most with no effect up to 1.0 U/ml Anti-Xa
• Variable LA effect
A quantitative fibrinogen is
extrapolated using the
clotting time obtained plotted
against a calibration curve
Fibrinogen: with concentrated thrombin reagent, no effect of UFH
up to 2.0 U/ml
Thrombin time: with dilute thrombin reagent, alternative test for
UFH or DTI monitoring in patients with elevated baseline (pretreatment) aPTT
Influence on coagulation testing
• Preanalytical
– In-vivo
– Ex-vivo
• Analytical
Preanalytical variables
• Blood sample
– Too little versus too much
– Too long
– Difficult phlebotomy
– 3.2% vs 3.8% buffered sodium citrate
– Venipuncture vs line draws
– Tube sequence??
– Unintended effects:
• Drugs
• Other nonpathologic events e.g. stress, diet,
oral contraceptives and other hormonal
changes, etc
Analytical variables
• Instrumentation
– optical versus mechanical clot detection
• Affected by interferences
– lipemia, bilirubinemia, etc.
• Reagents
– PT source (recombinant, brain extract,
etc)
– aPTT
• activator
• phospholipid source and concentration
• designed for heparin, factor VIII & IX, poss
LA
Possible causes of  INR and/or aPTT
Preanalytical
Short draw – excess sodium citrate in plasma
Elevated HCT (>55%) - common in neonates – excess citrate in plasma
Clotted sample
Sample >4 hours old: ↑ aPTT
Sample >24 hours old: ↑ INR
Hemodilution-drawn above IV site; drawn via arterial line without proper
clearance
Factor deficiencies
PT factors VII, X, V, II and to a lesser degree, fibrinogen
aPTT factors XII, XI, IX, VIII, X, V, II, and to a lesser degree, fibrinogen
Physiological decrease:
 Hereditary deficiencies: Factor VIII and IX most common, with
higher incidence of factor XI deficiencies in Azhkenazi Jewish
population
 Immature liver: premature infants and neonates
 Liver disease ↑ INR with normal or slight ↑ aPTT
Consumptive coagulopathy
Hemodilution
RBC transfusion without FFP
Blood volume expanders
Deficiencies can also be associated with antibody directed against
factors
Drugs
PT: Oral vitamin K antagonists
Daptomycin
Direct thrombin inhibitors
Argatroban > bivalirudin > lepirudin
Activated protein C (Xigris)
Systemic fibrinolytic activators (e.g. urokinase)
aPTT: Unfractionated heparin
Direct thrombin inhibitors
Activated protein C (Xigris)
Systemic fibrinolytic activators (e.g. urokinase)
Hydroxy-ethyl starch, hematin, sumatin, taularidine
Antiphospholipid antibodies – varies with reagent, may ↑ INR in addition to
aPTT
Possible causes of  INR and/or aPTT
Preanalytical
Low HCT (<25%)
Elevated Calcium levels
Poorly collected sample ↓ INR and/or aPTT due to
activated sample
Samples placed on ice ↓ INR
Sample > 2 hours for patients on UFH: ↓ aPTT due to
platelet degranulation and PF4 release, which
neutralizes heparin.
Elevated factor levels
Inflammatory response ↓ aPTT due to elevated factor VIII
and/or fibrinogen
Cryoprecipitate - contains high levels of factor VIII and
fibrinogen
Drugs
INR:
Activated factor VII (Novseven)
Prothrombin complexes (Proplex or Autoplex)
aPTT:
Prothrombin complexes (Proplex or Autoplex)
Direct factor therapy (Humate, Kogenate, Alphanine)
Summary
• Most coagulation screening tests are
crude in nature
• All coagulation results should be
interpreted with caution until preanalytical and analytical variables have
been excluded to prevent false
positive/negative results
• Good luck with validation process…