Laboratory Assessment and Selected Therapeutic Interventions in Hemostasis Flashcards
In vivo vs in vitro
In vivo - how it happens in the body…need to understnad for disease
In vitro - how it happens outside the body…important for understanding lab results
Platelet count
150-400 thousand/uL
Spontaneous bleeding risk low unless count is <10,000
Thrombotic risk at 1 million
Bleeding time
Standardized skin incision
Blot blood flow with filter paper until it comes away clean and measure time til it stops
Preop risk assessment for bleeding…not good
PFA-100
Superior to bleeding time
Citrated whole blood aspirated through hole in membrane lined with collagen plus epineprhine or ADP
Activated platelets aggregate
Measure time for platelet plug to occlude hole - closure time
Citrate tube sequesters calcium and only measures primar y
Prolonged CT
Thrombocytopenia, vWD, aspirin ingestion, Glanzman, thrombasthenia
not useful for antiplatelet drug monitoring
Good neg predictive value…normal CT means not primary hemostatic dz
PLatelet aggregation testing
Platelets concentrated into platelet rich plasma …various platelet activation facotrs added individually
Activation leads to GP2b/3a mediated aggregation…aggregated clumps fall out of suspension…measurable light transmission increases
Uses light transmission aggregometry
Impedence aggregometry
Whole blood…platelet aggregation increases electrical resistance
Glanzmann thrombasthenia on LTA
Will show flat line because GP2b/3a receptors don’t work and no aggregation
Bernard Soulier dz on LTA
Looks normal
Ristocetin must be used to diagnose B-S…binds to vWF and induces conformation change that exposes normally hidden GP1b binding domain, enabling it to agglutinate with platelets
Stoarge pool dz on LTA
Heterogenous
Aspirin will flatten the curve like Glanzman
Congenital aspirin like defect
Platelets do not respond to arachidonic acid
Aggregation vs agglutination
Aggregation - depends on GP2b/3a
Agglutination - depends on GP1b
Aspirin affect on platelets
Aspirin irreversibly inhibits platelet COX enzymnes and blocks TXA2 production…inihibits TXA2 mediated platelet activation
Transfers its acetyl group to the COX 1 enzyme
GP2B/3a antagonists
Phosphodiesterase inhibitors
GP2B/3a antagonists - block platelet aggregation
Phosphodiesterase inhibitors promote persistance of cyclic AMP and GMP, enhancing platelet inhibitory NO effects
THienopyridines
Block platelet activation (clopidogrel and prasugrel)
Permanently bind P2Y12 ADP receptors
VerifyNow
Rapid test for aspirin or plavix effect on patients
Employs fibrinogen coated beads whcih aggregate in proportion to GP2B/3A receptors (fewer exposed if more blocked by drug)
Agonist (ADP for plavix and AA for aspirin) activates platelets, exposing GP2B/3S which binds the beads
Ristocetin cofactor activity
Test for vWF (%)
Patients native platelets removed and replaced with formlain treated reagent platelets
Formalin treated will agglutinate at rate proportional to concentration
ELISA assay for vWF
Uses anti-vWF ABs to quantitate amount
Types of vWD and quantitation
1 and 3 - decreased vWF production…protein quantity and activity low
Type 2 - abnormal that may or may not be decreased…activity low but amount may be normal
RIPA
Ristocetin induced platelet aggregation - test for type 2B vWD
Should agglutinate at lower concentrations if type 2B because conformation already present
Test for type 2B
If diagnosed, can treat with DDAVP
Multimer analysis
Good for vWD
Type 1 and 3 - decrease in all sizes
Type 2 - selective loss of HMW multimers
TTP - increased multimer
Clot based test advantages and disadvantages
Well established, available, simple, cheap, familiar
Potentially misleading
Clot based tests
Being with platelet poor plasma…excess calcium neutralizes citrate…reagnet phospholipid fills role of removed platelets…clot formation stimulated with an agonist…end point is initial detection of clot
Only for secondary hemostatic dz
Endpoint of clot based tests
Fibrinogen to fibrin cleavage (detecting fibrin formation)
Where do in vitro and in vivo converge?
Factor 10
Common pathway sequecne
10, 2 ,1
Extrinsic pathway
Tissue factor, F7, F10, 2, 1
Intrinsic pathway
F12 forms with HMWK and PK…F12 cleaves F11…F11 cleaves F9 (needs F8)…cleaes F10…2, 1
PTT
PT
Isolated PTT (partial thromboplastin time) prolongation points to extrinsic Isolated PT (prothrombin time) pointed to F7
PTT
Partial thromboplastin time
All elements intrinsic to ciruclation (No TF)
TO start, silica added with Ca and phosphlipids removed
Phospholipids without TF - partial thromboplastin
Commonly used for monitoring unfractionated heparin
PT
Prothrombin time
Evaluates extrinsic pathway
TF needed to start the reaction…added with Ca and phospholipids previously removed
Phospholipids plus TF - thromboplastin
Common used for warfarin dosing
INR
International normalized ratio
Intended to standardize PT results across labs
Used for warfarin
Warfarin
Requires frequent labs
Dose affected by Vit K intake, drug effects of CYP2C9 metab, genetic polymorphisms of CYP2C9 and VKO reductase activity
If needed to start warfarin early for thromboembolism, must
Bridge with heparin due to short half life of F7…if started on full warfarin, would run out of protein C and become hypercoagulable…risk is warfarin induced skin necrosis
ISI
International sensitivity index - closer to 1 is better
INR formula
(PT patient/PT normal)^ISI
Range should be 2-3
PT and PTT mixing studies
How to tell if due to deficiency or inhibitor
If PT/PTT corrects into the normal range, then due to factor def
If not, due to inhibitor like LAC or factor specific AB
SPecific factor assays
Plasma deficienct in the specific factor of interest is added to patient speciemn…in absence of inhibitor, degree of prolongation inversely correlates with factor activity
Urea clot lysis
Used to test for F13 def
urea will lyse a clot of uncrosslinked…will not lyse clot of cross linked
Factor def
Single factor - mild hemophilia, if F12, then doesn’t matter
Multiple factors - liver dz or vit K def
Inhibitors
Heparin
LAC
Specific factor inhibitoras
In mixing study, if mix corrects
Need to obtain factor activity levels
If it doesn’t correct, rule out heparin exposure or contam, and consider LAC testing
Most factor specific antibodies at
Anti-F8 - bethesda unit score…put in human plasma until 50% F8 emerges…inverse is BUS
LAC testing
IN vitro clotting time prolonged
Mixing study does NOT correct
Add excess of phospholipid and if it corrects, then lupus anticoagulant and repeat in 8-12 weeks
DRRVT
Dilute Russell viper venom time
Tests for LA subtypes…anti-beta-2 glycoprotein…more specific for clinically signifant LA
What does DRVV do?
Acvtiates F10 to initiate clotting and bypasses both intrinsic and extrinsic pathways…If LAC present, prolongs clot time
ACA testing
ELISA based assay
Cardiolipin Ag bound to sides of test tube…binds, detected using anti-human-Ig ABs
Heparin
Liver, gut, basophils, mast cells
Heparin has super negative charge
Chemically similar to heparans found on endothelial cells walls
All heparins have a common pentasaccharide sequence that fits into AT
Heparin types
UFH - unfractionated heparin - range of molecular sizes with up to 30 additional sugars
LMWH - low molecular weight heparin - only smaller
Fondaparinux - only the pentasaccharide
Effect of heparin molecular size
Inhibition of thrombin requires a larger heparin molecule to span both other molecules
This is not necessary for F10 inhibition
Thus LMWH inhibits F10 but not thrombin
UFH affect
Prolongs PT and PTT
LMWH affect
Not reliably prolonging of PT and PTT
If needed to monitor - use anti-Xa assay
Anti-Xa assay
Add F10a to patient plasma…measure activity of F10…inversely proportional to heparin concentration
ACT
Advanced clotting time
Also a way to monitor heparin
Protamine
Binds and inactivates heparin
D-dimer values
Elevation demonstrates prior formation of cross linked fibrin
Expected to be elevated in VTE, surgery, trauma
Negative predictive value in reuling out VTE in case of PE
Rotational viscoelastography
Thromboelastography
Measures whole blood clot strength over time
Can help distinguish between anatomic and coagulopathic bleeding,
Can detect hypercoagulability to avert thrombosis
Indentifies fibrinolysis
Optimizes use of blood products and antifibrinolytic drugs
