Fundamentals of Coagulation Testing - PowerPoint PPT Presentation

1 / 41
About This Presentation
Title:

Fundamentals of Coagulation Testing

Description:

Fundamentals of Coagulation Testing Robert Gosselin, CLS University of California, Davis Health System Department of Clinical Pathology and Lab Medicine – PowerPoint PPT presentation

Number of Views:80
Avg rating:3.0/5.0
Slides: 42
Provided by: globalhea6
Category:

less

Transcript and Presenter's Notes

Title: Fundamentals of Coagulation Testing


1
Fundamentals of Coagulation Testing
  • Robert Gosselin, CLS
  • University of California, Davis Health System
  • Department of Clinical Pathology and Lab Medicine
  • Sacramento, CA
  • Robert.gosselin_at_ucdmc.ucdavis.edu

2
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

3
Goals and Objectives
  • Resident driven
  • What do you want?
  • What do you need?
  • Bringing questions to the table

4
Hemostasis
  • Cellular
  • Endothelium
  • Platelets
  • Red blood cells
  • White blood cells
  • Fluidic
  • Procoagulant factors and regulators
  • Fibrinolytic factors and regulators

5
Role of endothelium
  • Procoagulant
  • Collagen
  • vWF stored in WP bodies
  • Tissue factor expression
  • Cytokine/chemokine

6
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

7
Cytokines/Chemokines
  • TNF-a
  • IL1-ß
  • 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
  • C3aC4aC5a induce elastase release

TF expression
8
Cellular components
  • Platelets
  • Receptors
  • Adhesion
  • Aggregation
  • Factors
  • Intracellular
  • Adhesion
  • Procoagulant factors

9
AD Shapiro, WFH 199919
10
Role of platelets
TF
  • collagen exposure
  • vWF-GPIb-IX
  • GPIIb-IIIa
  • Release of ATP,ADP
    TF serotonin, B-TG Fbg,
  • Thrombin

P selectin exposure Monocyte adhesion
CR
Fbg
Platelet
IX
X
V
II
11
Cellular components
  • RBCs
  • Facilitate platelets to the endothelial surface
  • Thromboplastin source
  • WBCs
  • Tissue factor expression-MC
  • Cytokine expression
  • Elastase production-PMNs

12
Revised Cascade
Tissue Factor
VII
VIIa-TF
VIIa-TF
IX
X
XIa
IXa
neutralized
VIII
VIIIa
Xa
VIIa-TF-Xa
TFPI
V
Va
Prothrombin
Thrombin
F1.2
13
Cytokine release
THROMBIN
Anticoagulant
Procoagulant
Fibrinogen cleavage fibrin monomer generation
Complex with thrombomodulin protein C activation
Activates factor VIII and V
Generate TAFI fibrinolysis inhibitor
Induces platelet aggregation
Endothelial release of tPA
Activate factor XIII
14
Thrombin
XIII
Fibrinogen
Fibrin
XIIIa
FDP
Stabilized Fibrin
Plasminogen
uPA or tPA
Plasmin
uPAR
FDP
uPARuPA
CLOT LYSIS
MMP
D-dimer
Extracellular degradation
15
D
E
E
E
D
D
FIBRIN
D
D
D
D
tPA
D-dimer
Plasminogen
Plasmin
uPA
FIBRINOGEN
D
D
E
E
D
D
E
Fragment Y
Fragment X
D
Fragments D E
Fragment D
16
HMWK Pre
Traditional Waterfall Cascade
XII
XIIa
VII
XIa
XI
Ca2
TF
IXa
IX
VIIa
PF4 Ca2
VIIIa
VIII
Xa
X
PF4 Ca2
Va
V
Prothrombin
Thrombin
F1.2
Fibrin
Fibrinogen
FPA FPB
17
HMWK Pre ?
Primary Coagulation Regulators
XII
XIIa ? ?
AT ? PC ? PS ? C1 Inh ? HCFII ? TFPI ? ?2-MG ?
VII
XIa ? ?
XI
Ca2
TF
IXa ?
IX
VIIa ?
PF4 Ca2
VIII
VIIIa ? ?
Xa ? ?
X
PF4 Ca2
Va ? ?
V
Prothrombin
Thrombin ? ? ?
Fibrin
Fibrinogen
18
Thrombin
XIII
Fibrinogen
Fibrin ?
PAI-I? TAFI? ?2-AP? ?2-MG?
XIIIa
FDP
Stabilized Fibrin
Plasminogen
uPA or tPA ?
Plasmin
uPAR
???
FDP
uPARuPA
CLOT LYSIS
MMP
D-dimer
Extracellular degradation
19
Tests for endothelial function
  • Nada
  • All indirect measurements
  • Soluble factors
  • vWF, Endothelial-1
  • Cytokine

20
Test for WBC and RBC function
  • WBC
  • Indirect assessment
  • Soluble factors
  • Flow cytometry
  • Aggregates
  • Cytokine production
  • RBC
  • HCT

21
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

22
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)

23
Principle of the PFA-100
  • Collagen/Epinephrine (CEPI) primary screening
    cartridge
  • Collagen/ADP (CADP) differentiates dysfunction
    due to aspirin

Results reported as Closure time in seconds
24
Coagulation methods-functional
  • Clot detection
  • Screening versus specific
  • e.g. aPTT versus factor VIII
  • Light scatter vs mechanical
  • Chromogenic
  • Immunologic

25
Coagulation methods-antigenic
  • ELISA
  • Electrophoresis
  • Immunologic

26
Coagulation Testing in the Clinical Laboratory
  • Prothrombin times (PT)
  • PP Plasma Activator ( CaCl2) Clot
    Detection
  • 37oC
  • Activated partial thromboplastin times (aPTT)
  • PP Plasma Activators CaCl2
    Clot Detection
  • 37oC

Optical methods change in turbidity Mechnical
methods change in motion
27
Clot based tests
28
CRUMMY BLOOD DRAW Increased activation Delays in
testing ? PF4 release
? Factor levels Factor VIII
Fibrinogen Drugs Fxs DDAVP PCCs Novo7
Foods (e.g. caffeine) Exercise
(Physiological) Stress
29
  • ? Factor activity
  • Drugs- UFH, DTI, Xigris
  • Lipemia, icterus
  • /- Lupus anticoagulant
  • Inhibitors

Crummy blood draw Factor consumption Delays in
testing ? Fx VIII activity
30
Anti-Xa activity
plasma heparin exogenous antithrombin
Excess fXa
AT-heparin-Xa complex residual fXa
Chromogenic substrate
yellow color
31
HIT antibodies IgG ()
Conjugated Anti-human IgG antibody
Chromogenic tag ?
Wash
Wash
Microwell containing target antigen PF4-heparin
complex



Color



?
?
?









Amount of color proportional to amount of
antibody present
Amount of color proportional to amount of
antibody present
Incubate
Incubate
Reagent beads coated with anti-vWF
Patient vWF ?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
Instrument readingchanges in optical density
secondary to aggregates
?
?
Incubate
Testing well
32
Test Validation
33
HMWK Pre
XII
XIIa
XII
VII
XIa
XI
XI
Ca2
VII
IX
TF
IXa
IX
VIIa
VIII
PF4 Ca2
VIIIa
VIII
X
Xa
X
V
PF4 Ca2
Va
V
II
Prothrombin
Thrombin
Fibrin
Fibrinogen
aPTT
PT
34
aPTT
  • Screen for factor deficiencies
  • Monitoring drug effect
  • UFH
  • DTI
  • Factor VIII IX replacement Rx
  • Other
  • LA

35
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

36
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 (pre-treatment) aPTT
37
Influence on coagulation testing
  • Preanalytical
  • In-vivo
  • Ex-vivo
  • Analytical

38
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

39
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

40
(No Transcript)
41
Summary
  • Most coagulation screening tests are crude in
    nature
  • All coagulation results should be interpreted
    with caution until pre-analytical and analytical
    variables have been excluded to prevent false
    positive/negative results
  • Good luck with validation process
Write a Comment
User Comments (0)
About PowerShow.com