section 8 - testing methods

Question 1

list mechanisms of coagulation instrumentation

Answer 1

• mechanical
• photo-optical (turbidometric)
• nephelometric
• chromogenic
• immunologic

Question 2

describe mechanical clot detection

Answer 2

• electro: two probes, clot completes the circuit = stop timer
• magnetic: less movement of steel ball = breaks contact with magnetic sensor when clot forms

Question 3

describe photo-optical/turbidimetric principle of coagulation testing

Answer 3

measures change in optical density (light transmittance) through a sample
- less light = stop timer = clot formed

Question 4

describe nephelometric coagulation instrumentaitn

Answer 4

• antibody-antigen complex precipitates causing turbidity that scatters light

Question 5

describe chromogenic coagulation instrumentation

Answer 5

• based on color changing substance pNA
• protein protease frees pNA
• intensity is proportional to protease activity

Question 6

describe immunologic coagulation instrumentation

Answer 6

• antigen-antibody reactions w/ coated Ab microlatex
• particles agglutinate and absorb light equal to amount of Ag present

Question 7

what factors are measured in PT

Answer 7

• VII, X, V, II and I
• adequacy of the extrinsic pathway
• cannot measure the alternative pathway

Question 8

what is in the PT reagent

Answer 8

• thromboplastin: TFIII, PFIII and Ca
• can contain a heparin inhibitor

Question 9

reference range of PT

Answer 9

11-13 seconds

Question 10

how do oral anticoagulants impact PT

Answer 10

• used to monitor effects
• should have a PT of 20-27

Question 11

define INR

Answer 11

• international normalized ratio
• correction standardizes patient PT between laboratories
• measures PIVKA (vit K factors) effect on PT

Question 12

how does the ISI relate to INR

Answer 12

• 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)

Question 13

INR calculation and reference range

Answer 13

INR= (patient PT/normal PT) ^ ISI
- reference: 0.9-1.2 (not taking coumadin)
- therapeutic reference range: 2.0-3.0

Question 14

what does the APTT measure

Answer 14

• adequacy of intrinsic pathwaya

Question 15

how is APTT measured

Answer 15

• activator reagent for contact factors, PF3 and Ca
• activator reagent + PPP –(warmed)–> add Ca –> time to clot

Question 16

APTT reference range

Answer 16

26-36 seconds

Question 17

what is the APTT used for

Answer 17

intrinsic pathway adequacy and effects of heparin
- heparin patients = 1.5-2.5 times normal value

Question 18

describe the thrombin time test (TT)

Answer 18

• adequacy of fibrinogen
• reagent = dilute thrombin

Question 19

how is TT measured

Answer 19

PPP + 100 micro liters thrombin reagent

Question 20

TT reference range

Answer 20

14-20 seconds

Question 21

describe the purpose of a 50:50 mix

Answer 21

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

Question 22

what causes a prolonged PT/APTT or TT if a 50:50 mix corrects the time to clot

Answer 22

factor deficiency
- normal plasma is supplying the deficient factor

Question 23

what causes a prolonged PT/APTT or TT if a 50:50 test does not fix the prolonged time

Answer 23

due to inhibitor
- inhibitor also inhibits normal plasm

Question 24

describe the immunological fibrinogen assay

Answer 24

• RID or nephelometry
• quantitates total fibrinogen including dysfunctional

Question 25

describe the fibrinogen activity assay

Answer 25

- mechanical or photo-op method - quantitates **functional fibrinogen** - most common assay

Question 26

describe the fibrinogen assay

Answer 26

- 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

Question 27

reagents in fibrinogen assay

Answer 27

- fibrinogen calibration reference - thrombin - owrens veronal buffer (diluent)

Question 28

reference range for fibrinogen assay

Answer 28

200-450 mg/dL

Question 29

describe D-Dimer assay

Answer 29

- present only in fibrinolysis - all assays use monoclonal Ab - ELISA/ELFA and latex agglutinattion most common -> OD, fluorescence or visible agglutination

Question 30

D-Dimer interpretation

Answer 30

- if no agglutination test is reported as <0.5 ug/mL never 0 - see in cases of active thrombosis -> DIC -> DVT -> PE

Question 31

describe the priciniple of the factor assay

Answer 31

- 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

Question 32

what is the % activity of factor

Answer 32

using dilutions of normal plasma and deficient plasma see how active the factor is - read % activity off of the curve

Question 33

reference range for factor acvitity

Answer 33

60-150%

Question 34

describe the anti-Xa assay

Answer 34

- 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

Question 35

describe the russell viper venom time (RVVT)

Answer 35

- venom is a direct activator of factor X - measures common pathway - reference range 20-30 seconds

Question 36

RVVT reference range

Answer 36

20-30 seconds

Question 37

describe the reptilase time test

Answer 37

- 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

Question 38

reference range of reptilase time (RT)

Answer 38

16-22 seconds

Question 39

what is the issue if TT is prolonged but RT is normal

Answer 39

thrombin inhibitor, not fibrinogen deficiency

Question 40

what is the issue if TT and RT is prolonged

Answer 40

fibrinogen deficiency or FDPs present in high amounts

Question 41

describe the Urea Clot lysis (urea solubility)

Answer 41

- 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

Question 42

describe the euglobulin clot lysis test

Answer 42

- qualitative measure of pt endogenous fibrinolytic capability - monitors fibrinolytic activity of tPA medicine pt - check fibrinogenolysis amount

Question 43

euglobulin clot lysis procedure

Answer 43

- 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

Question 44

euglobulin clot lysis reference range

Answer 44

>1 hour (usually less than 4 hours) - increased fibrinolytic activity = clot lysis in <1 hour

Question 45

describe activated clotting time (ACT)

Answer 45

- whole blood clotting time - point of care assay - monitors high dosage heparin during surgery - intrinsic pathway through contact factors