PowerPoint: Coagulation Automation

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Coagulation Automation

Joanna Ellis, MLS (ASCP)

Keri Brophy
-
Martinez,
MHA/ED (ACHE), MT(ASCP)



Screening Tests


Bleeding Time


Manual method that evaluates primary
hemostasis (being replaced by PFAs)


Prothrombin time (PT)


Extrinsic and Common Pathways


Activated Partial Thromboplastin Time (aPTT)


Intrinsic and Common Pathways


Thrombin Time (TT or TCT)


Conversion of Fibrinogen to Fibrin


Quantitative Fibrinogen


Determines amount of fibrinogen


D
-
Dimer


Detects fragments from plasmin degradation of
the fibrin clot






Specialized Tests



Platelet Aggregation studies


Measures ability of VWF to support agglutination
of normal platelets by ristocetin


Platelet Function Assay (PFA)


Tests platelet adhesion and aggregation


Thrombelastography (TEG)


Real
-
time view of all stages of hemostasis


Mixing Studies



Identifies specific factor deficiencies or inhibitors


Specific Coagulation Factors



Determines actual activity of a factor such as
Factor VIII or IX


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


Automation approaches to clot
detection:


SEE


Turbidometric


Nephelometric


FEEL


Mechanical


Viscosity based



Optical Clot Detection
(Turbidimetry)


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


Nephelometric Clot
Detection



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



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.

Viscosity Based Clot
Detection



Sample added to a cuvette with a wire or
steel ball


The steel ball or wire swings like a pendulum
between two electromagnetic fields on the
sides of the cuvette.


As the plasma clots, the viscosity decreases
the swinging motion of the wire or ball.


The variation in the amplitude of this
swinging motion is used to determine the
clotting time

Tests that use a Clot
Detection Method



PT


APTT


TT


Fibrinogen


Mixing Studies


Specific Coagulation Factor Assays


FVIII


FIX


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


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.


Aggregating Reagents

(Agonist)



Collagen


ADP


Epinephrine


Ristocetin


Arachidonic Acid

Platelet Function Analyzer

(PFA
-
100)


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)


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


VWF:Ristocetin Cofactor

VWF:RCo



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.




Platelet aggregation patterns
in various disorders



Thrombelastography (TEG
®
)


Whole Blood based analysis


Monitors hemostasis in its entirety


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 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


Reduces unnecessary use of blood products


Allows effective management of hypercoaguability


Differentiates surgical from pathological bleeding

References


"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.discovery
-
diagnostics.com/Cascade_M4.asp>.


"Fiche Produit
-

Stago."
Homepage Stago Corporate
-

Stago
. Web. 14 Nov.
2010. <http://www.stago.com/nc/products
-
services/catalogue/analyzers/fiche
-
produit/selection/type
-
analyzers/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/servlet/ProductDi
splay~q_catalogId~e_
-
111~a_catTree~e_100001,1023065,1028378,1015818~a_langId~e_
-
111~a_productId~e_182047~a_storeId~e_10001.htm>.