section 8 - testing methods Flashcards
list mechanisms of coagulation instrumentation
- mechanical
- photo-optical (turbidometric)
- nephelometric
- chromogenic
- immunologic
describe mechanical clot detection
- electro: two probes, clot completes the circuit = stop timer
- magnetic: less movement of steel ball = breaks contact with magnetic sensor when clot forms
describe photo-optical/turbidimetric principle of coagulation testing
measures change in optical density (light transmittance) through a sample
- less light = stop timer = clot formed
describe nephelometric coagulation instrumentaitn
- antibody-antigen complex precipitates causing turbidity that scatters light
describe chromogenic coagulation instrumentation
- based on color changing substance pNA
- protein protease frees pNA
- intensity is proportional to protease activity
describe immunologic coagulation instrumentation
- antigen-antibody reactions w/ coated Ab microlatex
- particles agglutinate and absorb light equal to amount of Ag present
what factors are measured in PT
- VII, X, V, II and I
- adequacy of the extrinsic pathway
- cannot measure the alternative pathway
what is in the PT reagent
- thromboplastin: TFIII, PFIII and Ca
- can contain a heparin inhibitor
reference range of PT
11-13 seconds
how do oral anticoagulants impact PT
- used to monitor effects
- should have a PT of 20-27
define INR
- international normalized ratio
- correction standardizes patient PT between laboratories
- measures PIVKA (vit K factors) effect on PT
how does the ISI relate to INR
- ISI is the defined sensitivity of individual reagents
- reference thromboplastin has a value of 1
- further ISI of a reagent is from 1, the less sensitive to PIVKA (vit K nonfunctional factors)
INR calculation and reference range
INR= (patient PT/normal PT) ^ ISI
- reference: 0.9-1.2 (not taking coumadin)
- therapeutic reference range: 2.0-3.0
what does the APTT measure
- adequacy of intrinsic pathwaya
how is APTT measured
- activator reagent for contact factors, PF3 and Ca
- activator reagent + PPP –(warmed)–> add Ca –> time to clot
APTT reference range
26-36 seconds
what is the APTT used for
intrinsic pathway adequacy and effects of heparin
- heparin patients = 1.5-2.5 times normal value
describe the thrombin time test (TT)
- adequacy of fibrinogen
- reagent = dilute thrombin
how is TT measured
PPP + 100 micro liters thrombin reagent
TT reference range
14-20 seconds
describe the purpose of a 50:50 mix
if PT, APTT or TT is prolonged = it determines if it is due to deficiency or inhibitor
- normal plasma and patient plasma in a 1:1 ratio
what causes a prolonged PT/APTT or TT if a 50:50 mix corrects the time to clot
factor deficiency
- normal plasma is supplying the deficient factor
what causes a prolonged PT/APTT or TT if a 50:50 test does not fix the prolonged time
due to inhibitor
- inhibitor also inhibits normal plasm
describe the immunological fibrinogen assay
- RID or nephelometry
- quantitates total fibrinogen including dysfunctional
describe the fibrinogen activity assay
- mechanical or photo-op method
- quantitates functional fibrinogen
- most common assay
describe the fibrinogen assay
- quantitates fibrinogen activity only functional fibrinogen
- similar to TT but plasma diluted 1:10 w/ thrombin added each time
- used standard curve to convert mg/dL of fibrinogen
reagents in fibrinogen assay
- fibrinogen calibration reference
- thrombin
- owrens veronal buffer (diluent)
reference range for fibrinogen assay
200-450 mg/dL
describe D-Dimer assay
- present only in fibrinolysis
- all assays use monoclonal Ab
- ELISA/ELFA and latex agglutinattion most common -> OD, fluorescence or visible agglutination
D-Dimer interpretation
- if no agglutination test is reported as <0.5 ug/mL never 0
- see in cases of active thrombosis
-> DIC
-> DVT
-> PE
describe the priciniple of the factor assay
- used when PT or APTT is prolonged to determine which factor is missing
- specific factor deficient plasma and pt plasma
-> if remains prolonged = deficient
-> if fixed = not the problem
what is the % activity of factor
using dilutions of normal plasma and deficient plasma see how active the factor is
- read % activity off of the curve
reference range for factor acvitity
60-150%
describe the anti-Xa assay
- monitors LMW heparin, UFH or direct Xa inhibitors
- chromogenic assay
- add pt plasma + reagent (constant Xa) + chromogenic = degree of color is inversely proportional to amt AT in sample
describe the russell viper venom time (RVVT)
- venom is a direct activator of factor X
- measures common pathway
- reference range 20-30 seconds
RVVT reference range
20-30 seconds
describe the reptilase time test
- reptilase cleaves only fibrinopeptide A from fibrinogen (enough to start clotting)
- not inhibited by thrombin inhibitors (heparin)
- differentiates deficiency of fibrinogen from inhibitor
- RR: 16-22 seconds
reference range of reptilase time (RT)
16-22 seconds
what is the issue if TT is prolonged but RT is normal
thrombin inhibitor, not fibrinogen deficiency
what is the issue if TT and RT is prolonged
fibrinogen deficiency or FDPs present in high amounts
describe the Urea Clot lysis (urea solubility)
- determines adequacy of FXIII
- clot formed with PRP and Ca in glass tube -> 5 M urea added -> see if clot dissolves
- if clot dissolves = inadequate FXIII
describe the euglobulin clot lysis test
- qualitative measure of pt endogenous fibrinolytic capability
- monitors fibrinolytic activity of tPA medicine pt
- check fibrinogenolysis amount
euglobulin clot lysis procedure
- euglobulin = plasminogen, plasmin/plasmin activators and fibrinogen
- euglobulin fraction precipitated in acetic acid
- precipitate recovered and resolubilized in buffer
- fibrinogen is added to clot
- watch for clot lysis
euglobulin clot lysis reference range
> 1 hour (usually less than 4 hours)
- increased fibrinolytic activity = clot lysis in <1 hour
describe activated clotting time (ACT)
- whole blood clotting time
- point of care assay
- monitors high dosage heparin during surgery
- intrinsic pathway through contact factors
