Section 8: Hemostasis Testing Flashcards
If hct is low, do you need more or less anticoagulant?
- More anticoag
- Low hct means more plasma
If hct is high, do you need more or less anticoagulant?
- Less anticoag
- High hct means less plasma
What type of sample do coag studies vs platelet studies use?
- Coag studies use PPP
- Plt studies use PRP
Coag specimens should always be handled and prepared in what kind of material?
Plastic to prevent contact activation by glass
Prothrombin Time holding sample conditions
- room temp up to 24 hr
- FVII gets activated at colder temps
Plasma sample holding time conditions
- -20C for 2 wk
- -70C for longer than 2 wk
- freeze/thaw rapidly at 37C or ice crystals denature proteins
APTT holding sample conditions
- up to 4 hrs
- if on heparin centrifuged within 1 hr and tested within 4 hr
describe manual tilt tube method
- add reagents and specimens to tube and maintain heat by heat block
- time how long it takes for plasma to clot
when would you use manual tilt tube method?
- sometimes pt plasma won’t get turbid when it clots, and automated analyzers measure light transmittance change when a clot forms, so false neg
- so do manual method to troubleshoot
Electromechanical clot detection (mechanical)
- measures the change in conductivity between 2 metal probes immersed in plasma
- when fibrin clot forms it completes the previously unclosed circuit and stops the timer
Magnetically monitoring clot (mechanical)
- as clot forms, test solution increases in viscosity
- detects the change in movement of the steel ball by changing its range of motion or a break in contact with magnetic sensors
Instrument we’re using in coag lab
Diagnostica Stago STArt 4
Photo-optical (turbometric) coag testing
- measures change in OD of test sample
- light of a specific wavelength passes thru sample. As clot forms, the plasma becomes more opaque and decreases light detected
Nephelometric coag testing
- immunometric method form measuring proteins
- antigen-Ab complexes ppt -> turbidity-> scatters light
- detects variations in light scatter at 90° and 180°
- if immune complexes are too small then Ab are attached to latex particles
Chromogenic (amidolytic) coag instrumentation
- based on chromophore usually para-nitroaniline (pNA)
- goal is for coagulation protein (protease) to attack chromogenic substrate to free pNA
- free pNA is yellow. Color intensity proportional to amount of protease activity
Immunologic endpoint coag testing
- based on antigen-Ab interactions
- microlatex particles coated with specific Ab to analyte
- Beam of monochromatic light passes thru suspension
- wavelength of light greater than diameter of individual particles, so only small amount of light absorbed
- When Ag-Ab complexes form, their bigger diameter allows more light absorption
PT tests for what pathway? List factors
- Extrinsic
- FVII, X, V, II, I
Sample and reagents for PT?
- sample is PPP in sodium citrate
- thromboplastin (mix of phospholipids + TF3 + CaCl2)
PT procedure
- warm thromboplastin to 37°C
- PPP warmed to 37°C separate tube
- add thromboplastin to pt specimen and observe for clot formation
- time recorded for clot formation is the PT
PT cannot test for what?
- TFIII, Ca2+, PF3
- XIII
PT reference range
11-13 sec
PT used for what? What should the PT be?
- monitoring effects of oral anticoags
- these pt should have PT of 20-27 s
international normalized ratio (INR)
- standardizes pt results between labs
- commercial reagents are assigned a sensitivity index by WHO - thromboplastin is assigned value of 1
- Farther ISI from 1, the less sensitive it is to PIVKAs
PIVKAs
proteins induced by Vit K absence or antagonists
INR calculation
INR = (Pt PT/Normal PT)^ISI
INR ref range
0.9-1.2
APTT tests for what pathway? List coag factors
- intrinsic
- XII, XI, IX, VIII, PK, HMWK
Contact factors
XI, XII, PK, HMWK
APTT reagents
- activator reagent made of contact activators and phospholipid (PF3 sub)
- CaCl2
APTT procedure
- mix activator reagent with pt PPP
- warm mixture at 37°C. XIII activates during inc
- add prewarmed CaCl2 to initiate clotting
- time required to clot is the APTT
APTT cannot test for what?
- PF3 or Ca2+
- XIII
APTT ref range
26-36 sec
APTT used to monitor what? What should APTT results be?
- heparin effects
- 1.5-2.5 times normal value
If both PT and APTT are prolonged, which are possible single factor deficiencies?
X
V
II
I
common pathway
TT procedure
- dilute thrombin is warmed to 37°C
- warm pt PPP to 37°C in separate tube
- add prewarmed thrombin to pt tube
- time required to clot is the TT
thrombin time (TT)
- tests for adequacy of fibrinogen
- dilute thrombin is reagent
- only measures last step of coag cascade
TT ref range
14-20 sec
when to perform 50:50 mix “corrected TT”
do if PT, APTT, or TT is prolonged in order to find out if it’s due to a factor deficiency or an inhibitor
50:50 mix result if abnormal test is due to inhibitor
Test should remain prolonged since the inhibitor will also inhibit the normal plasma
fibrinogen assays that measure total fibrinogen, even dysfunctional
- RID method
- nephelometry
fibrinogen assays that measure only functional fibrinogen
mechanical or photo-optical method
fibrinogen assay (fibrinogen functional activity) based on same principle as TT with two exceptions. What are they?
- plasma diluted 1:10 used to minimize effects of inhibitors (e.g., heparin)
- the results of the test are reported in mg/dl using a standard curve to convert clot time to concentration
fibrinogen assay reagents
- fibrinogen calibration reference for standard curve
- thrombin
- owrens veronal buffer diluent
fibrinogen assay standard curve procedure
- several dilutions of the fibrinogen calibration reference are made. Each warmed 37°C
- thrombin is added to each dilution and the clotting time is recorded
- on log-log graph paper, the fibrinogen level of each dilution is plotted against its clotting time
- multiply fibrinogen conc of each dilution by 10 so that pt results can be directly read off graph
fibrinogen assay patient and control procedure
- dilute specimens 1:10 with veronal buffer
- 0.2 ml of diluted specimen is warmed to 37°C
- add 0.1 ml of thrombin, record the time it takes for a clot to form
- compare this time to the standard curve and determine mg/dl of fibrinogen
Why reduce the effects of inhibitors in fibrinogen assay?
Inhibitors can interfere with the normal coagulation process, leading to inaccurate measurements of fibrinogen levels. By minimizing their effects, the assay can more accurately reflect the true fibrinogen concentration
fibrinogen assay ref range
200-450 mg/dl
how are d-dimers formed?
- fibrin polymers stabilized by XIIIa to form covalent cross-linkages in D domain to make insoluble clot
- when plasmin breaks down fibrin clot it makes two D fragments held together by XIIIa, aka D-dimer
- D-dimers only present in fibrinolysis
List immunoassays that test for D-dimer ( all use mAb)
- ELISA
- immunofluorescence
- latex agglutination
- turbidometric
- nephelometry
- whole blood agglutination
describe whole blood agglutination for D-dimer testing
bi-specific antibody attaches to RBC Ag at one end and then attaches to D-dimer at the other end, leading to agglutination
D-dimer seen in which cases of active thrombosis?
DIC
DVT
PE
D-dimer beware of
postzone phenomenon
principle of factor assay
- use specific factor deficient plasma to determine which factor is deficient
- APTT remains prolonged if have VIII deficiency and use VIII deficient plasma (50:50 mix)
- if deficient in some other factor, the APTT will correct
how to find patient’s % activity of specific factor
- prepare standard curve with factor deficient plasma mixed with normal plasma and make dilutions
run APTT on dilutions and plot seconds vs % activity (can also use PT) - run APTT on 1:5 and 1:10 dilutions of pt plasma + factor VIII deficient plasma
factor assay reference range
60-150%
when to do anti-Xa assay
to monitor therapy with LMWH, UFH, or direct factor Xa inhibitors
anti-Xa assay procedure
- patient plasma + reagent w constant amount Xa
- add chromogenic substrate
- increase color means less LMWH, less color means more LMWH (more inhibition, more anti-thrombotic)
Russell viper venom time (RVVT)
- snake venom from the russell pit viper is a direct activator of factor X
- mixing venom with calcium and PPP tests the common pathway
RVVT ref range
20-30 s
reptilase time (RT)
- snake venom from Bothrops Atox acts like thrombin does except it only cleaves fibrinopeptide A, still enough to start clotting process
- reptilase not inhibited by common thrombin inhibitors such as heparin and auto-Ab
how to use RT
differentiate a lack of fibrinogen from the presence of an inhibitor
what can inhibit RT?
FDPs can inhibit RT but to lesser degree
TT prolonged and RT normal
what’s going on?
thrombin inhibitor present bc it doesn’t inhibit reptilase, but it does inhibit thrombin
TT prolonged and RT prolonged
what’s going on?
- FDP inhibitors
- lack of fibrinogen
urea clot lysis (urea solubility) purpose
determine adequacy of XIII
urea clot lysis procedure
- form clot with PRP and calcium in glass tube. If pt lacks enough fibrinogen then can add exogenous fibrinogen
- add 5M urea or 1% monochloroacetic acid
- incubate at 37°C for 24 hr
- if clot is stabilized by enough XIII activity, the clot will remain. If not adequate, the clot will dissolve
euglobulin clot lysis used to do what?
- qualitatively measure the patient’s endogenous fibrinolytic capability
- monitor pts getting tPA, streptokinase, or urokinase therapy
- make sure pt is not experiencing dangerous degree of fibrinogenolysis as result of therapy
euglobulin clot lysis procedure
- euglobulin (aka fibrinolytic component) of plasma is precipitated using 1% acetic acid
- ppt recovered by centrifugation and resolubilized in buffer
- the fibrinogen is clotted by adding thrombin (may add exogenous fibrinogen if pt level <200 mg/dl
- incubate mixture at 37° and observe every 10 min for clot lysis
euglobulin clot lysis procedure ref range
> 1hr
what’s in the euglobulin fraction of plasma?
plasminogen
plasmin
plasmin activators
fibrinogen
Activated clotting time (ACT)
- whole blood clotting time
- clot activator in tube to speed process
- widely used as point of care test to monitor high dosage therapy during cardiac catheterization or coronary artery bypass surgery
