Coagulation AutomationCoagulation Automation

Joanna Ellis, MLS (ASCP)

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

Screening TestsScreening 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 TestsSpecialized 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)/RoTEM◦ 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 InstrumentationCurrent Instrumentation

To See or To FeelTo See or To Feel

Automation approaches to clot detection:◦SEE Turbidometric Nephelometric

◦FEEL Mechanical/Viscosity based

Instrument Instrument MetholodologiesMetholodologiesOptical/TurbidometricNephelometricMechanicalChromogenicImmunologic

Optical Clot Detection Optical Clot Detection (Turbidimetry)(Turbidimetry)

Sample is added to a cuvetteA light source is directed through the

cuvette Initial absorbance of transmitted

light is measuredClot initiating reagents are added by

the automated instrumentationThe 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-4Cascade M-4(Instruments at RRC)(Instruments at RRC)Four cuvettes can be analyzed

at one timeSemi-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

Nephelometric Clot Nephelometric Clot DetectionDetection

Sample is added to a sample cuvetteThe optically clear cuvette passes in

front of light sourceThe clot initiating reagents are

addedLight is scattered as the fibrin

strands formThe light scatters at different angles

and is measured by detectorsA clot curve is generated by

consecutive readings until clot completion.

Mechanical Clot Mechanical Clot DetectionDetection

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 KC1 (Instruments at EVC)(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 DetectionChromogenic DetectionUses a colorless substrate and a

chromophoreProtease activity of the factor allows the

substrate-chromophore complex to be cutColor change results and the OD is measured

at 405 nm.

Allows for specific coagulation factor activity to be measured.

Immunologic Light Immunologic Light AbsorbanceAbsorbanceUses latex particles coated with antibodies

against select antigensOnce 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 Tests that use a Clot Detection MethodDetection Method

PTAPTTTTFibrinogenMixing StudiesSpecific Coagulation Factor

Assays◦FVIII◦FIX

Aggregating ReagentsAggregating Reagents(Agonist)(Agonist)

CollagenADPEpinephrineRistocetinArachidonic Acid

Platelet Function AnalyzerPlatelet Function Analyzer(PFA-100)(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 Platelet Function AnalyzerAnalyzer(PFA-100)(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 Platelet AggregometryAggregometry

Performed in specialized labs by experienced laboratory professionals

Performed on Aggregometer utilizing photometry

Measures light transmittance over a period of time

Platelet aggregation patterns Platelet aggregation patterns in various disordersin various disorders

VWF:Ristocetin VWF:Ristocetin CofactorCofactorVWF:RCoVWF: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.

Thrombelastography Thrombelastography (TEG(TEG®®))Citrated Whole Blood based

analysisMonitors 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 Thrombelastography (TEG(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 ResultTEG Graphic Result

Typical TEG Graph Typical TEG Graph PatternsPatterns

Uses of TEGUses 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

productsAllows effective management of

hypercoaguabilityDifferentiates surgical from

pathological bleeding

RoTEMRoTEMRotational Rotational thromboelastometrythromboelastometry

Similar to TEGUses a heated cup that remains

stationary, while pin oscillates as the clot forms◦Uses automated pipetting

Provides data on clot kineticsUses optical detectionAdvantage- less sensitive to agitation

(compared to TEG)

ReferencesReferences "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>.

ReferencesReferences 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/ProductDisplay~q_catalogId~e_-111~a_catTree~e_100001,1023065,1028378,1015818~a_langId~e_-111~a_productId~e_182047~a_storeId~e_10001.htm>.