Coagulation Automation Joanna Ellis, MLS (ASCP) Keri Brophy-Martinez, MHA/ED (ACHE), MT(ASCP)

Screening Tests 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)/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 Instrumentation

To See or To Feel Automation approaches to clot detection: ◦ SEE  Turbidometric  Nephelometric ◦ FEEL  Mechanical/Viscosity based

Instrument Metholodologies Optical/Turbidometric Nephelometric Mechanical Chromogenic Immunologic

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

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

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.

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 substrate- chromophore complex to be cut Color change results and the OD is measured at 405 nm. Allows for specific coagulation factor activity to be measured.

Immunologic Light Absorbance 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 PT APTT TT Fibrinogen Mixing Studies Specific Coagulation Factor Assays ◦ FVIII ◦ FIX

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.

Platelet Aggregometry Performed in specialized labs by experienced laboratory professionals Performed on Aggregometer utilizing photometry Measures light transmittance over a period of time

Platelet aggregation patterns in various disorders

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.

Thrombelastography (TEG ® ) Citrated 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 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 ◦ Reduces unnecessary use of blood products Allows effective management of hypercoaguability Differentiates surgical from pathological bleeding

RoTEM Rotational thromboelastometry Similar to TEG Uses a heated cup that remains stationary, while pin oscillates as the clot forms ◦ Uses automated pipetting Provides data on clot kinetics Uses optical detection Advantage- less sensitive to agitation (compared to TEG)

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 Web. 14 Nov "Fiche Produit - Stago." Homepage Stago Corporate - Stago. Web. 14 Nov "KC1 DELTA COAG ANALYZER 1/EA - Trinity Biotech # G05000." LabSource.com - Your Source for Science and Safety! Web. 14 Nov

References McGlinchey, Kevin. "» More on Trinity’s KC1 and KC4 Educational Promotion." The Fritsma Factor: Your Interactive Hemostasis Resource. 5 Nov Web. 14 Nov McGlinchey, Kevin. "Coagulation Automation." Advance 19.6 (2010): Print. McKenzie, Shirlyn B. "Chapter 40." Clinical Laboratory Hematology. 2nd ed. Boston: Pearson, Web. "PFA-100® System." Siemens Healthcare Worldwide Web. 14 Nov