Laboratory Evaluation of Hemostasis Flashcards
petechiae and mucus membrane bleeding are signs of…
PLT/vascular defect (primary hemostasis)
ecchymosis, bleeding into joints, and hematuria are signs of…
coagulation defects (secondary hemostasis)
specimen requirements and handling for evaluation of hemostasis
- clean venipuncture (avoid trauma; no TF release; prevent activation of clotting factors
- avoid EDTA, heparin, and fluoride contamination (collect coag tubes first)
- use anticoagulant (3.2% sodium citrate)
> reversibly binds free calcium in sample
> preserves factors V and VIII best
> does not inhibit PLT function - clotted specimens useless; invert 4-5 times only
ratio of anticoagulant to blood in citrate tubes must be
1: 9
* *tubes must be filled completely when being collected**
- if too little = overanticoagulation and testing times will be falsely prolonged
- using butterfly needle = discard tube must be collected first (accounts for air vol in tubing)
if severe hemolysis occurs, the release of this occurs
erythrocytin
T or F. Factors V and VIII are stable
F! they are labile!! if not tested right away, remove plasma and freeze
bleeding disorders (3 categories)
- vascular and PLT disorders
- coag factor defs/abnormalities
- fibrinolytic disorders
tests for PLT/vascular disorders (4)
- PLT count (automatic methods usually; impedance or flow cytometry)
- bleeding time (not done anymore)
- PLT aggregation tests
- Ristocetin cofactor tests
this detects qualitative abnormalities in patients who have a normal PLT count
PLT function tests
Platelet aggregometry/light transmission aggregometry
- continuous measurement of light transmitted through a suspension of PLTs in plasma ; more aggregation = more light transmittance
- variety of agonists to activate aggregation = thrombin, ADP, arachidonic acid, epinephrine, collagen, ristocetin
- each agonist has a typical transmittance pattern
T or F. 0% Transmittance = 0% aggregation
T!
antibiotic that cases vWF to bind GP Ib/IX/V on platelets
Ristocetin
- agglutination (not true aggregation)
- requires presence of vWF (ristocetin cofactor) and GP Ib/IX/V
PLT Aggregation Test interpretation of results
- Glanzmann Thrombasthenia: GPIIb/IIIa def; abnormal aggregation w all agonists except ristocetin
- Von Willebrand Disease: normal aggregation with all except ristocetin
- Bernard-Soulier syndrome: GPIb def; same as vW disease
Ristocetin cofactor test
- differentiate between vW disease and Bernard-Soullier syndrome
- done when PLT aggregometry is abnormal with ristocetin
- repeat aggregometry using:
> ristocetin
> normal plts: ensures GPIb/IX/V is present
> patient’s plasma: must provide vWF - still abnormal = vWF disease 9patient’s plasma did NOT provide vWF)
- normal = Bernard-Soulier disease
confirmatory testing for ristocetin cofactor test
vWF antigen asay or GP Ib, IX, V receptors
factor abnormalities can be caused by:
- decreased synthesis
- production of dysfuncitonal coag factor molecule
- excessive destruction of factors through acquired disorders
- inactivation of factors through circulating inhibitors
tests for coag factor abnormalities
- prothrombin time
- activate partial thromboplastin time
- thrombin time
- screening test for circulating inhibitors
- fibrinogen assay
- single facto assays
- factor XIII screen/factor XIII assay
clot-based testing
- measures time from initiation of clotting to clot formation
> endpt = clot
> prolonged clotting time indicates coagulopathy
Endpt clot detection methods: (2)
- visual (manual): time stos when a clot is seen
- automated
> optical endpt - change in absorbance
> mechanical - measures viscosity
> electrical circuit completed as a clot forms
prothrombin time (PT)
- screens for factor deficiecies in extrinsic and common pathway; presence of circulating inhibitors
> factors VII, X, V, prothrombin and fibrinogen - measures time for clot to form when reagent containing tissue thromboplstin, phospholipids, and Ca 2+ are added to plasma = activates VII => common pathway
- also used to monitor oral anticoagulant therapy (warfarin/Coumadin)
PT is reported as …
international normalized ratio (INR)
- corrects for variability in thromboplastin used
- PT results around the world are standardized; critical for monitoring patients on Coumadin
PT is reported as …
international normalized ratio (INR)
- corrects for variability in thromboplastin used
- PT results around the world are standardized; critical for monitoring patients on Coumadin
aPTT
- screens for factor deficiencies and inhibitors in the intrinsic and common pathways
> prolong in defs of PK, HMWK, XII, XI, IX, VIII, V, prothrombin, fibrinogen - plasma incubated with reagent that contains activator and phospholipid
- contact factors activated and XIa formed
- addition of Ca 2+ allows progression to formation of fibrin clot
- used to monitor heparin anticoag therapy - ref range varies with each lot # of reagent
inhibitor screen
- normal plasma added to patient plasma to see if there s a correction of a prolonged PT/PTT result
> corrected = factor def (normal plasma provides missing factor)
> no correction = inhibitor is present (inhibits factors in both plasmas) - additional test to: determine type and specificity of inhibitor + identify factor that is deficient
- inhibitors can be against specific factors, or non-specific
thrombin time
- measures time needed for thrombin to convert fibrinogen to a fibrin clot
> directly activates fibrinogen to bypass previous steps in coagulation pathways - uses dilute thrombin reagent
- detects deficient/defective fibrinogen and thrombin inhibitors [heparin and fibrin degradation products (FDPs)]
- also prolonged due to oral direct thrombin inhibitor therapy
thrombin time
- measures time needed for thrombin to convert fibrinogen to a fibrin clot
> directly activates fibrinogen to bypass previous steps in coagulation pathways - uses dilute thrombin reagent
- detects deficient/defective fibrinogen and thrombin inhibitors [heparin and fibrin degradation products (FDPs)]
- also prolonged due to oral direct thrombin inhibitor therapy
thrombin time
- measures time needed for thrombin to convert fibrinogen to a fibrin clot
> directly activates fibrinogen to bypass previous steps in coagulation pathways - uses dilute thrombin reagent
- detects deficient/defective fibrinogen and thrombin inhibitors [heparin and fibrin degradation products (FDPs)]
- also prolonged due to oral direct thrombin inhibitor therapy
fibrinogen assay
- measures clotting when exces thrombin is added
> clotting time inverself proportional to amount of fibrinogen - modification of thrombin time procedure = excess thrombin neutralizes heparin and FDPs
- results are read from a standard graph
> prepared by testing known concentrations of fibrinogen
single factor assays (ex: FVIII)
- patient plasma is added to factor deficient plasma and tested using PT or PTTT
- measure ability of patient plasma to correct factor def plasma
> patient plasma provides deficient factor
> results are compared to normal plasma
T or F. Factor XIII deficiency is not detected by PT or PTTT
T!
- endpt is before fibrin crosslinking
- factor XIII stabilizes fibrin clot by converting hydrogen bonds into covalent bonds
- measures time for a fibrin clot to disolve in 5M urea or 1% monochloroacetic acid
- if XIII def, clot dissolves <1 hr in monochloroacetic acid or <24 hrs in 5M urea
- factor XIII assay performed to confirm deficiency/identify functional abnormalities
