Transcript Coagulation Automation

Joanna Ellis, MLS (ASCP)
Keri Brophy-Martinez, MHA/ED (ACHE),
MT(ASCP)
Coagulation Automation
Screening Tests
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Bleeding Time
◦ Manual method that evaluates primary
hemostasis (being replaced by PFAs)
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Prothrombin time (PT)
◦ Extrinsic and Common Pathways
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Activated Partial Thromboplastin Time (aPTT)
◦ Intrinsic and Common Pathways
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Thrombin Time (TT or TCT)
◦ Conversion of Fibrinogen to Fibrin
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Quantitative Fibrinogen
◦ Determines amount of fibrinogen
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D-Dimer
◦ Detects fragments from plasmin
degradation of the fibrin clot
Specialized Tests
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Platelet Aggregation studies
◦ Measures ability of VWF to support agglutination of normal platelets by
ristocetin
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Platelet Function Assay (PFA)
◦ Tests platelet adhesion and aggregation
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Thrombelastography (TEG)/RoTEM
◦ Real-time view of all stages of hemostasis
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Mixing Studies
◦ Identifies specific factor deficiencies or inhibitors
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Specific Coagulation Factors
◦ Determines actual activity of a factor such as Factor VIII or IX
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Antithrombin (AT or ATIII)
◦ In the presence of heparin, low levels of AT indicate poor clinical
response to heparin
Current Instrumentation
To See or To Feel
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Automation approaches to clot
detection:
◦ SEE
 Turbidometric
 Nephelometric
◦ FEEL
 Mechanical/Viscosity based
Instrument Metholodologies
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Optical/Turbidometric
Nephelometric
Mechanical
Chromogenic
Immunologic
Optical Clot Detection
(Turbidimetry)
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Sample is added to a cuvette
A light source is directed through the
cuvette
Initial absorbance of transmitted light is
measured
Clot initiating reagents are added by the
automated instrumentation
The plasma becomes more opaque when
clotting is initiated, decreasing the light
transmitted through the cuvette
The change in transmitted light is used to
calculate the result
Cascade M-4
(Instruments at RRC)
Four cuvettes can be analyzed at one
time
 Semi-automated Optical Clot
Detection
◦ The technician delivers the sample
and reagents into the cuvette
◦ The changes in optical density are
monitored
◦ Clot times determined by
instrument
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Nephelometric Clot
Detection
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Sample is added to a sample cuvette
The optically clear cuvette passes in
front of light source
The clot initiating reagents are added
Light is scattered as the fibrin strands
form
The light scatters at different angles and
is measured by detectors
A clot curve is generated by consecutive
readings until clot completion.
Mechanical Clot Detection
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The sample is introduced to a cuvette that has a
small steel ball inside
The cuvette continuously moves when testing
begins
The clot initiating reagents are added to the
sample
The fibrin strands begin to form and attach to
the moving ball
An electrical circuit is either opened or closed
when the ball moves away from the magnet
because of the fibrin strands
Clot time is recorded.
KC1
(Instruments at EVC)
•Semi-Automated Mechanical Clot
Detection:
•The Ball Method uses a steel ball at the
bottom of a cuvette that is held in place by
a magnetic source.
•While the cuvette continuously rotates,
the technician adds the sample and
reagents, which starts the timer. T
•When true clot formation has occurred,
the clot will incorporate the steel ball and
pull it away from the magnetic source,
stopping the timer.
Chromogenic Detection
Uses a colorless substrate and a chromophore
 Protease activity of the factor allows the substratechromophore complex to be cut
 Color change results and the OD is measured at 405
nm.
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Allows for specific coagulation factor activity to be
measured.
Immunologic Light Absorbance
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Uses latex particles coated with antibodies against
select antigens
Once latex particles and antigens agglutinate, more light
is absorbed by the forming clot.
An increase in light absorbance is proportional to the
antigen level.
Tests that use a Clot
Detection Method
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PT
APTT
TT
Fibrinogen
Mixing Studies
Specific Coagulation Factor Assays
◦ FVIII
◦ FIX
Aggregating Reagents
(Agonist)
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Collagen
ADP
Epinephrine
Ristocetin
Arachidonic Acid
Platelet Function Analyzer
(PFA-100)
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Uses stimulators of platelet adhesion and
aggregation in an environment that
stimulates an injured blood vessel wall.
More sensitive screening test than the
bleeding time method
Offers increased sensitivity for platelet
dysfunction and von Willebrand’s disease
Nonspecific test- not diagnostic for any
single disorder
Platelet Function Analyzer
(PFA-100)
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The instrument adds citrated blood to a
reservoir with either collagen/epinephrine
(EPI) or collagen/adenosine diphosphate
(ADP) on a bioactive membrane
A pressure sensor detects the formation of
a platelet plug on the membrane
The time it takes to close the aperture in
the membrane with the platelet plug is
recorded.
The result is a function of platelet count,
platelet activity,VWF activity, and
hematocrit.
http://www.platelet-research.org/3/pfa.htm
Platelet Aggregometry
Performed in specialized labs by
experienced laboratory
professionals
 Performed on Aggregometer
utilizing photometry
 Measures light transmittance
over a period of time
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Platelet aggregation patterns in
various disorders
VWF:Ristocetin Cofactor
VWF:RCo
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Slowly centrifuged citrate sample yields
platelet-rich plasma (PRP).
The PRP must be adjusted with the patients
PPP to reach a standard number of 200,000/µL
The sample is stirred, warmed to 37°C in a
photometric aggregometer
The aggregating reagent (agonist) is added
◦ In this case, Ristocetin
The platelets begin to aggregate which leads
to a change in optical density (OD) of the PRP
as measured by a absorbance detector.
The aggregometer records the changes in OD
in a graphic curve.
Thrombelastography (TEG®)
Citrated Whole Blood based analysis
 Monitors hemostasis in its entirety
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◦ Clot initiation through clot lysis
◦ Measures the net effect of all hemostatic
components interacting together during the
clotting process
◦ Demonstrates the hemostatic potential of a
blood sample at a given point in time.
Thrombelastography (TEG®)
•Sample of citrated whole blood is
placed in a cup which has a pin carefully
connected to a torsion wire.
•As the cup rotates in a back and forth
movement, the aggregates formed within
the cup cause the wire to become more
rigidly placed and reflects the strength
of the aggregates formed within the
cup.
•The movement or lack of movement is
reflected via either an optical or
magnetic detector
•A graphic presentation is produced
TEG Graphic Result
Typical TEG Graph Patterns
Uses of TEG®
Illustrates function and dysfunction in the
Hemostatic system
 Allows physicians to give appropriate
amounts of FFP, Cryo, and platelets to
control hemorrhage
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◦ Reduces unnecessary use of blood products
Allows effective management of
hypercoaguability
 Differentiates surgical from pathological
bleeding
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RoTEM
Rotational thromboelastometry
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Similar to TEG
Uses a heated cup that remains stationary,
while pin oscillates as the clot forms
◦ Uses automated pipetting
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Provides data on clot kinetics
Uses optical detection
Advantage- less sensitive to agitation
(compared to TEG)
References
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"Cascade M M4 Hemostasis Coagulation Analyzers Clotting Assays
PTs APTTs Thrombins Fibrinogens Factor Assays - Discovery
Diagnostics Canadian Distributor Helena Laboratories." Hematology
Stainers Microbiology Stainers Cytocentrifuges Osmometers Sweat
Collection Blood Temperature Indicators Fecal Occult Blood Platelet
Aggregation. Discovery Diagnostics and JLS Web Designs, 8 Sept.
2008. Web. 14 Nov. 2010. <http://www.discoverydiagnostics.com/Cascade_M4.asp>.
"Fiche Produit - Stago." Homepage Stago Corporate - Stago. Web. 14
Nov. 2010. <http://www.stago.com/nc/productsservices/catalogue/analyzers/fiche-produit/selection/typeanalyzers/reference/58609/group/sta-compact/>.
"KC1 DELTA COAG ANALYZER 1/EA - Trinity Biotech # G05000."
LabSource.com - Your Source for Science and Safety! 2009. Web. 14
Nov. 2010.
<http://www.labsource.com/Catalog/Item.aspx?ItemID=1392043>.
References
McGlinchey, Kevin. "» More on Trinity’s KC1 and KC4
Educational Promotion." The Fritsma Factor:Your Interactive
Hemostasis Resource. 5 Nov. 2009. Web. 14 Nov. 2010.
<http://www.fritsmafactor.com/newfritsmafactor/?p=2044>.
 McGlinchey, Kevin. "Coagulation Automation." Advance 19.6
(2010): 26-27. Print.
 McKenzie, Shirlyn B. "Chapter 40." Clinical Laboratory
Hematology. 2nd ed. Boston: Pearson, 2010. Web.
 "PFA-100® System." Siemens Healthcare Worldwide. 2007. Web.
14 Nov. 2010.
<http://www.medical.siemens.com/webapp/wcs/stores/servle
t/ProductDisplay~q_catalogId~e_111~a_catTree~e_100001,1023065,1028378,1015818~a_lan
gId~e_111~a_productId~e_182047~a_storeId~e_10001.htm>.
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