Week 8 Flashcards

Question

how do you test for PT

Answer 1

PT reagent - source of Tissue Factor or Thromboplastin or Tissue Thromboplastin ACL Elite PT reagent - RecombiPlasTin® Recombinant Thromboplastin - commercially prepared Requires reconstitution using diluent Suspended in phospholipids Mixed with buffered solution of calcium chloride (source of ionized Ca2+) -PT reagent warmed to 37 -PPP warmed to 37 for 3-10 mins anything longer coag factors degrade, or are affected by evaporation or pH -add reagent quickly -clot forms, timer stops and time in secs noted -clot detected visually or by electrical sensors ranges are determined by the lab

Answer 2

-Activates the extrinsic and common pathways -Begins with FVII to Fibrin polymerization = Fibrin clot formation -commercial reagent has Ca and phospolipids that also partake in the reaction to form -TF:FVIIa complex (extrinsic tenase complex) -Factor FXa:Va complex -Thrombin activation, fibrin polymerization, and fibrin clot formation

Answer 3

APTT – clot-based routine coagulation test -used to assessfactors in the intrinsic and common pathway: Factors XII, XI, IX, VIII, X, V, II (prothrombin), I (fibrinogen) – all factors except FVII -monitor standard (unfractionated) Heparin anticoagulant therapy

Answer 4

-Alcohol (can slow dow breakdown of warfarin - causing a buildup in the body) - antibiotics (increase PT/INR) -Barbiturates, oral contraceptives, and hormone-replacement therapy (HRT) (decrease PT or prothrombotic) -Foods that contain large amounts of vitamin K can interfere with Warfarin therapy -pt history is important and a bleeding/thrombosis risk assessment is done by the doctor

Answer 5

-International Normalized Ratio (INR) -allows for monitoring of Vitamin K antagonist therapy (such as, Coumadin/Warfarin) across different labs -INR with PT result ISI = International Sensitivity Index and PT in sec are used to calculate INR -ISI shows how sensitive the reagent is to V K dependent factors it is compared to WHO ref standard INR= pt PT/mean PT (from 20 normal plasma samples) to the power of ISI of the reagent used

Answer 6

-heparin therapy -detect Specific anticoagulation factor antibodies (i.e., Lupus Anticoagulant and/or anti-factor VIII antibody) or non-specific inhibitors - detect Coagulation factor deficiencies -Determine cause of Unexplained bleeding or easy bruising -Determine cause of recurrent miscarriages -Determine if Disseminated Intravascular Coagulation (DIC) is present -Determine reason behind recurrent thrombosis -Prior to surgery when the surgery carries an increased risk of blood loss and/or when the person has a clinical history of bleeding

Answer 7

-Congenital deficiency of FVIII, IX, or XI (usually a single factor deficiency) -Def of FVIII & IX associated with bleeding vWF disease has low FVIII and is associated with bleeding -Def FXI variably associated with bleeding -Contact factor def can elevate APTT but does not result in bleeding tendency Acquired inhibitors, specific or non-specific -Specific inhibitors directed against specific factors (commonly against FVIII) -Non-specific inhibitors can be drugs (heparin, rivaroxaban, apixaban, or edoxaban) or antiphospholipid antibodies (lupus anticoagulants) -Patients on Direct Thrombin Inhibitors

Answer 8

Reagent 1: Contact activator Contact activator (negatively charged particulate activator, such as, silica, ellagic acid, or kaolin) Phospholipids (PL) with NO TF or Ca2+ A ‘partial thromboplastin’ because it lacks Tissue Factor ACL Elite: SynthASil® Reagent 2: Calcium chloride CaCl2 is added separately ACL Elite: Calcium chloride

Answer 9

-PPP + R1 (contact activator + PL ) incubate for 3 min at37 Contact factor group activates FXI-FXIa which activates FIX-FIXa. Contact factor group= FXIIa + pre-K + HMWK. Intrinsic factor activation. -R2 (CaCl2) added, mix at 37, start timing in sec The Ca allows FIXa and FVIIIa to bind with phospholipid producing the Intrinsic tenase complex IXa:VIIIa + PL + Ca2+ that forms the Prothrombinase complex, Thrombin activation and clot formation. -stop timing when you see fibrin strands -stop fully when you or electrical sensors see a clot each lab has their own reference values -PT on heparin are 1.5-2.5X mean so multiply 1.5 for low end of mean and 2.5 for high end -ranges must be re-established once a year

Answer 10

Drugs: Antihistamines, ascorbic acid, chlorpromazine, heparin, and/or salicylates Incorrect anticoagulant Hematocrit that is markedly increased or decreased Blood sample from heparin lock or heparinized catheter

Answer 11

UFH therapy- use a reagent that is not sensitive to UFH one that has a UFH neutralizer like a polybrene LUPUS Anticoag- PT and PTT results are invalid use a chromogenic assay instead Incorrect calibration, dilution of reagents -correct the error and repeat

Answer 12

if coagulation test results appear short (or are more rapid) than RI’s, we do not investigate as they are not clinically significant. -Was sample collected with correct anticoagulant & in a properly filled tube? -ratio okay? -Clotted / Hemolyzed / Lipemic / Icteric? Clotted - means coagulation factors are all used up and we cannot test them Hemolyzed/Lipemic/Icteric - may affect ability to measure clot on some analyzers and can falsely shorten or prolong testing Check patient HCT ≥ 0.55 L/L? -redraw and adjust anticoag volume used due to skew blood:plasma ratio = more RBC than plasma

Answer 13

-compare with last results -pf on Anticoag therapy? Need to reason for prolonged result if so release -if not is there a diagnosis example liver disease affects PT then PT/APTT, V K PT/APTT affected -if history or diagnosis correlates with result then release

Answer 14

investigate if patient has: Decreased fibrinogen levels Specific single or multiple factor deficiency Inhibitor or antibody to factors Inhibitor (non-specific) or antibody interfering with test. because pt with SLE develop AB that affect APTT in vitro due to the incubation time (the time is enough for AB to neutralize PL in reagent) -DO REFLEX AND ADDITIONAL TESTING Thrombin Time Fibrinogen D-Dimer Mixing Studies Inhibitor testing Factor Assays

Answer 15

-assess deficiencies of fibrinogen or the presence of an inhibitor to thrombin (FIIa) -referred to as Thrombin Clotting Time (TCT) Recall, Thrombin polymerizes fibrinogen to form fibrin by cleaving fibrinopeptides A and B from plasma fibrinogen to form a detectable fibrin polymer Qualitative test This test bypasses all other factors and assesses the activity of fibrinogen The less fibrinogen available the longer it takes to form a clot

Answer 16

Congenital Fibrinogen deficiency -Decreased fibrinogen level (quantitative - -hypofibrinogenemia or afibrinogenemia) -Abnormal fibrinogen function (qualitative - dysfibrinogenemia) Acquired by Heparin therapy, direct thrombin inhibitor therapy or contamination Heparin contamination of a blood sample is suspected because samples are drawn through central line (heparin neutralization procedures are now used more commonly) Acquired via Presence of an inhibitor of thrombin (FIIa) or fibrinogen Acquired hypofibrinogenemia Liver Disease Massive hemorrhage Abnormal fibrinolysis increased products of clot breakdown like in DIC that affect fibrin polymerization

Answer 17

Bleeding or thrombotic episodes Recurrent miscarriages PT and APTT are unexpectedly both prolonged. Do TT to directly assess Fibrinogen function

Answer 18

PPP and reagent warmed at 37 -add reagent to PPP-Commercially prepared bovine Thrombin reagent . once mixed the thrombin will degrade -start time with Thrombin addition and stop at clot -test is Qualitative we only measure the time it took for the clot to form not how much fibrinogen is there or how much Fibrin is produced -Thrombin time is most sensitive to fibrinogen and presence of inhibitors of thrombin (Factor IIa). -Specimen errors that affect the PT and APTT also affect the TT.

Answer 19

Modification of the TT - estimates the concentration of functional fibrinogen Measures the rate of conversion of fibrinogen to fibrin in a diluted sample with of excess thrombin Clotting time correlates with levels of active fibrinogen that is present (inverse relationship)

Answer 20

Acquired hypofibrinogenemia -Bleeding episodes (consumption of fibrinogen) -Consumption such as with traumatic injury or DIC (hyperfibrinolysis) -Decrease production seen in severe Liver Disease Assess inflammatory conditions Fibrinogen is an acute phase reactant and can be non-specifically increased during acute or chron inflammation Recurrent miscarriages Suspected congenital fibrinogen deficiency or abnormality Hypofibrinogenemia (rare congenital decreased FIB levels) Dysfibrinogenemia (dysfunctional FIB) PT and APTT test are both unexpectedly prolonged when physician wants to evaluate pt risk of developing cardiovascular disease

Answer 21

-ppp + bovine thrombin is prewarmed at 37 -PPP diluted with buffer like Owren buffer 1:10 (calibration curve determined with normal plasma) -reagent added with concen of thrombin where thrombin cleaves fibrinogen to fibrin. So we are trying to see if there is any activity if Fibrinogen is at a low level -measure time in seconds and compare with calibration curve. Time and Fib = inverse

Answer 22

For low fibrinogen, a lower dilution may be needed For high fibrinogen a higher dilution may be needed In either case, result(s) are multiplied by the dilution factor to produce the correct result curve is produced by the owren buffer dilutions

Answer 23

-specific product of digestion of cross-linked fibrin ONLY -Marker of Thrombosis and Fibrinolysis -breakdown products of fibrin clot digestion by Plasmin -Plasmin cleaves fibrin, producing fibrin degradation products/fibrin fragments, like D-Dimer -Ddimer has two D domain fragments from fibrin monomers, that are crosslinked by FXIIIa as clot forms

Answer 24

-increased D Dimer indicated clot formation and breakdown -not specific to thrombosis Deep vein thrombosis (DVT) Pulmonary embolism (PE) Recent surgery or trauma cancers Acute or chronic infections or inflammatory diseases DIC Very heavy menstruation Normal Pregnancy - levels rise in normal pregnancy however, very high levels are associated with complications Healthy elderly patient

Answer 25

-negative D-Dimer used to rule out the diagnosis of venousthromboembolism . So if there are no clot being formed or broken down then the pt does not have a thrombotic condition - positive or elevated D-Dimer alone is not diagnostic of DIC, DVT or PE. They are non specific when elevated or positive -

Answer 26

-test depend on site and pt population they go from qual, to semi qaul to quanti. can be manual or automated - D-Dimer Latex Reagent - polystyrene latex particles coated with a monoclonal antibody highly specific for the D-Dimer domain (epitope) -ACL mixes control or pt plasma with Latex and Buffer = agglutination means Ddimers are present Agglutination is measured by a decrease in transmitted light at 405nm Degree of agglutination is directly proportional to concentration of D-Dimer in sample

Answer 27

Positive (agglutination) or Negative(no agglutination) if the test is used for a qualitative assessment only In quantitative or semi-quantitative measurements, D-Dimer reported in D-Dimer Units (DDU) or Fibrinogen Equivalent Units (FEU)

Answer 28

Slide Latex Immunoassay Procedure -needs a pos and neg control with Reagent 3/4 -mix R1 with pt sample and rock the slide like Pathodx -view agglutination

Answer 29

Endpoint detection - testing resulting in a finite endpoint and for routine coagulation testing this is clot formation Mechanical Photo-optical (turbidometric) Nephelometric - our ACLs use this method Chromogenic (amidolytic) Immunologic Viscoelastic

Answer 30

-uses magnetic sensor that monitors the movement of a steel ball within the test plasma NOT AFFECTED by plasma

Answer 31

Electromechanical I -as fibrin clot forms , vicosity of test plasma and reagent increases slowing ball movement -when the movement stops , timer stops = clotting time Electromechanical II -steel ball is put in inclined well and its position is detected by magnetic sensor -the well rotates but the ball stays put -when the fibron clot forms the ball is moved from position indicating clotting time

Answer 32

used by automated and semi auto coag instruments to detect clots -Measures the change in plasma optical density (OD or light transmittance) during clotting (Turbidimetric analysis) -Formation of fibrin strands causes the light to scatter = less arriving at the detector -as clot get denser the more light is scattered OD increase -A baseline OD is determined based on the color and clarity of the sample -Baseline OD is subtracted from final OD – this minimizes the effects of icterus or lipemic samples

Answer 33

-side scatter or forward-angle light scatter is measured -A light-emitting diode produces incident light at a 600 nm -A photodetector detects variations in light scatter at 90 degrees (side scatter) or at 180 degrees (forward-angle scatter) -As fibrin polymers form, side scatter or forward-angle scatter rise -timer stops when scatter reaches a predetermined density -Continuous readings throughout and a clot curve is produced

Answer 34

-Measures specific coagulation factor activity, by exploiting Factors’ enzymatic (protease) properties -colorless, synthetic substrate is attached to a chromophore like a protease structure -Activated Factors cleaves the chromogenic substrate and OD of solution is measured at 405nm -OD of solution proportional to protease activity -chromophore is released and measured at a photodetector at 405nm - BILIRUN WILL AFFECT THE RANGE -THEY ARE BOTH YELLOW -optical detection of the solution is proportional to protease activity - direct chromogenic assay -Indirect is also used

Answer 35

-Based on Ab-Ag reaction with coagulation antigen -latex coated AB against AG -Monochromatic light of a greater wavelength passes through the suspension of latex micro-particles and absorbed light is measured -Antigens - coagulation factors and proteins, such as D-Dimer -after agglutination light is absorbed because the particles have wavelength larger than the light -The absorbance is proportional to the agglutinate size which is proportional to the antigen level

Answer 36

Used for whole blood clotting - Thromboelastography Provides information on: Time to clot Kinetics of whole blood clot formation Clot strength Fibrinolytic activity

Answer 37

-as fibrinogen specimen converts to fibrin and a solid clot, the change in light transmission through the specimen is recorded. baseline-recorded before clotting occurs acceleration phase-reflects clotting and is normally steep because clotting is rapid. deceleration phase-represents the decreasing rate of clot formation as all available fibrinogen converts to fibrin. end-point-flat and stable, reflecting consumption of all fibrinogen. -absorbance on the y axis gives the time interval of when the clot was detected

Answer 38

-preparation of lyophilized controls and reagents -make sure volumteric pipette is chip free -no foam production -dont use contaminated water -calibrate analyzer

Week 8 Flashcards

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