Week 10 Flashcards
What is coagulation
formation of a blood clot AFTER injury to blood vessel
What is thrombosis
formation of a blood clot inside a intact blood vessel which obstructs the flow of blood through the circulatory system
What is embolus
when a portion of a large clot breaks free and travels around the body
What are the 4 manifestations of thrombosis
DVT
pulmonary PE
Stroke
MI
What are some types of VTE
Venous Thromboembolic Events
DVT- in calf or leg, in deep veins, caused by loss of mobility like illness or long flights
-pain, swelling, redness and heat
-Erythema and edema
Pe Pulmonary emboli
-Blood clot in pulmonary artery blocking blood flow to lung caused by a portion of a DVT clot that has travelled up to the lung - FATAL due to ischemia of lung tissue
-symptoms are similar to pneumonia
How is DVT examined
Ultrasonography
What is Arterial Thrombosis
-Formation of atherosclerotic plaque on walls of small arteries
-if plaques are unstable they rupture causing wall damage and platelet activation
-pts take aspirin to prevent AT
-Ebolisms to organs originates in heart or large arteries and then occludes arterioles, coronary arteries and carotid arteries
What can Arterial Thrombosis cause
Stroke:
-clot blocks brain vessel resulting in ischemia and loss of brain function
-most strokes are caused by blockage of carotid arteries
Coronary Artery Disease
-blot blocks coronary artery
-thrombi that block the coronary arteries result in MI
What are some risk factors to thrombosis
Acquired - lifestyle
Secondary - systemic disease
Congenital - inherited
What is thrombophilia
hypercoaguble state
-increases the risk of thrombosis
- caused by acquired and congential factors
- needs a combination of risk factors before thrombosis (clot formation) occurs
What are the three main factors that predispose to thrombosis and make up the virchows triad
Endothelial injury (BV wall damage)
- causing PLT activation
-TF exposure
-athersclerosis , catheter
Abnormal blood flow
-venostasis
-when plts are brought in contact with endothelium due to flow of blood
-bed rest, paralysis, A fib
Hypercoagulability/reduced inhibition
-increase of coag factor activation so there are more procoag factors in the plasma or a reduction of inhibitory factors of coag pathway
Acquired Thrombosis Risk Factors
Age/Immobility - after 50 your risk doubles due to abnormal blood flow
Diet/Lipid metabolism - eating fatty foods, not enough folate , high chol, high LDL leads to plaque formation
-BV wall damage
Elevated estrogen - pregnancy / birth control increases risk 4x
-stimulation of coag factors
post op trauma - DIC , endothial injury
Smoking - depends on degree - endothlial injury and abnormal blood flow
Inflammation - endo injury increases coag factors
Acquired Thrombosis Risk Factors
Secondary associated with systemic risk factors
Chronic inflammation /autoimmune disorder- lupus
Hepatic and Renal disorders - decreased production of inhibitory factors (hypercoagulability) or excretion of factors through urine
Myeloproliferative disorders
Essential thrombocytopemia ET
Polycythemia vera PCV
Leukemia - increases risk for DIC. Some tumors release TF which activate coagulation
Lupus SLE
-inflammatory and autoimmune disease
-produces Anti DNA, ACAPN, Lupus anticoagulant LAC - which targets phospholipids
-varied manifestations
-tissue damage from AB and complement fixing
-more common in women
-recurrent miscarriages and butterfly rash
how to detect Lupus in LAB
ANA- ana nuclear AB test - GOLD
-only some lupus pts have LAC , it can also be detected in RA
-LA causes prolonged APTT and normal PT
-LA is an AB to protein - phospholipid complexes
What is Lupus Anticoagulant or Antiphospholipid Syndrome APS
not all pts with SLE develop SLE some get APS
-complication from anti phospolipid AB
-IgM or IgG AB to phospholipid
-promote coagulation in vivo but anticoagulant in VITRO
-prolonged phospholipid dependent tests- PTT
-associated with SLE
Antiphospholipid Syndrome (APS) – Clinical Criteria:
Thrombosis = - venous and arterial at the same time
-pregnancy loss- 3 or more unexplained (through impaired nitric oxide release, atherosclerotic plaque development, promotes clotting, TF expression, increased Thromboxane -increase PLT)
-slight hemolytic anemia
-thrombocytopenia
Laboratory Criteria for LA / Antiphospholipid Syndrome (APS)
initial PTT is prolonged with LAC presence
but PTT can be prolonged due to Factor deficiency , inhibitors (non specific LAC), Anticoagulants
differentiate between these by doing mixing studies
How to do a mixing study
Normal plasma with sample plasma 1:1 ratio
Corrects- factor deficiency or factor inhibitor
Not Correct - non specific inhibitor like LAC - DRVVT -assay for lupus anticoagulant
if corrects then make new 1:1 and incubate
corrects- factor deficiency - do factor assays
Not correct - specific factor inhibitor - do factor inhibitor assays
What is drvvt
dilute russel viper venom test
-initiates coag cascade at FX (intrinsic) not affected by inhibitors or factor deficiencies of FXI, XII, IX or VII (intrinsic)
If a prolonged time is seen with drvvt then its likely due to anti phospholipid antibodies like LAC
Laboratory Criteria for LA / APS
if you suspect an antiphospholipid AB then you need to confirm with
dRVV or PTT with high phospholipid conc
the high phospholipid concentration neutralizes the AB so the dRVVT is corrected to normal through the AB:AG reaction
confirm with immunoassay
ACAPN
ANTI B1 glycoprotein AB
What are the criteria for the diagnosis of LA
1-prolonged phospholipid dependent clot formation when using an assay like low phospholipid PTT or DRVVT
2-failed to correct the prolonged clot time when mixed with normal platelet poor control plasma
3-correction of prolonged screen with the addition of excess phospholipids
4-exclusion of coagulopathies
DIC
-starts with excessive clotting (small clots form throughout bloodstream and block blood vessels) caused by a substance that enters the blood
part of disease process
complication of child birth
surgery or trauma
toxins - venom, TSS
Coag pathway will be activated and excess circulating thrombin will activate PLTs, catalyze fibrinogen to fibrin
The fibrinolytic pathway is activated due to excess clot formation but in DIC the fibrin will FAIL to polymerize and circulate freely instead therefore the plasmin response will be delayed
DIC pathophysiology
normal anticoagulant and fibrinolytic systems are overwhelmed and coagulation activation cant be contained
fibrin microthrombi occlude small vessels causing PLT, coag factor and protein consumption
Red cells are fragmented as they travel through blood vessels
-Fibrin degradation products or D dimers are generated
-leads to excessive bleeding which depletes PLTs and clotting factors needed to control bleeding
Diagnosis of DIC
Type of MAHA
-thrombocytopenia
-shistocytes on PBS
prolonged PT and PTT
increased D dimer
fibrinogen is decreased because fibrinolysis via plasmin is occuring
D dimer
Negative D dimer means its NOT DIC, PE or DVT
-Normal is <0.5 mg/L
- in DIC levels are 10000 - 20000 ng/L or 10mg/L
in PE or DVT they are a bit lower
What tests could you do for testing of D dimer
-latex fixation slide test (Qualitative)
-ELISA -turbidimetric with decease of transmitted light at 405; change is plotted on a standard curve to establish the concentration if D dimer
1)If D-dimers are present, they will agglutinate the latex particles > Clumps
2)If D-dimer absent, solution remains clear
What Conditions Resulting in Increased Risk of Thrombosis (inheritied )
Deficiencies in:
Antithrombin
Protein C
Free PS
TPA
Mutations of:
Factor V
F V Leiden (APCR)
Prothrombin
G20210A
Fibrinogen
dysfibrinogenemia
Plasminogen
PAI-1 (elevation)
order a thrombosis panel to narrow down what is causing the issue
most common are VENOUS thrombosis AT, PC, PS, APC-R
Antithrombin (AT) Deficiency:
lab assessment
congenital deficiency
-serine protease inhibitor that neutralizes thrombin
acquired in prolong heparin therapy, DIC, Liver disease etc
most cases are due to reduced production or due to mutations that cause abnormalities in protease binding site or heparin binding site
Lab assessment :assays, Clot based and chromogenic AT assay , immunoassay for AT concentration
AT Chromogenic Functional Assay:
pt AT binds heparin and is activated
AT + HEP bind to reagent FXa
chromogenic substrate is added but it will be hydrolyzed by the excess FXa not bound to AP+HEP
the color produced is inversely proportional to amount of AT
Immunoassay for AT Concentration
consists of a reagent made up of latex microbeads coated with monoclonal AB directed at AT
the AT in pt plasma binds the beads causing latex agglutination and increases turbidity
AT concertation is directly proportional to rate of light ABs change
Chromogenic vs Immunoassay for AT
Fuctional (chromogenic)
detects both quantitative (amount present) and qualitative (ability to bind protease) AT deficiencies
Immunoassay
Detects only quantitative AT (amount present)
DOESNT TEST FOR FUNCTIONALILITY
Inherited Protein C or S Deficiency
Thrombin binds Thrombomodulin which activates Protein C (APC). APC binds free Protein S to stabilize APC-S complex. This complex will go inactivate FVa and FVIIIa
Therefore, without Protein C or Protein S, NO inhibition of FVa and FVIIIa, so clotting continues
Protein C def and Protein S deficiency - are Vit K dependent (DIC, Liver Disease)
No complexes formed
no inhibition of FVa or FVIIIa
do chromogenic or clot based assays
enzyme immunoassay
Chromogenic Protein C Assay
1)Patient Plasma is mixed with Reagent containing Snake Venom (which is activator of Protein C)
2)Second reagent containing colour substance is added
3)APC in patient plasma will hydrolyze the substrate and create colour
Intensity of color is proportional to the activity of Protein C
Clot-Based Protein C Assay
1)Patent plasma is mixed with Protein C-depleted Normal Plasma
—This ensures normal levels of all factors EXCEPT Protein C
2) Reagent containing snake venom and APTT reagent is added
3) Time to clot is measured
Prolongation is proportional to plasma protein C activity (because APC will prolong PTT)
APC prolongs APTT
Chromogenic
Clot based
both can affect molecule’s serine protease site (site which binds to FVIIIa and FVa)
**listen
Enzyme Immunoassay for Pro C
Uses ELISA method – Quantitative and Qualitative
-Plate coated with monoclonal Ab directed to Pro C. These monoclonal Ab are used to capture patient’s plasma Pro C
-Add an enzyme linked monoclonal antibody (also directed to Pro C)
-Add a substrate that changes colour when in contact with that enzyme
-Concentration of Pro C is measured by colour development
-Assay determines if low functional activity is due to non-functional Pro C (Qualitative) or if there is a quantitative deficiency (Quantitative – normal levels, dysfunctional protein)
Protein S Assays
Clot-Based Assay
Immunoassay
Pt plasma + protein S depleted normal plasma +reagent (APC, venom, and PTT)
time to clot is measured
prolonged = high protein S- proportional
Immunoassay - similar to Prc assay for qualitative and quantitative
Factor V Leiden (FVL) Mutation
Activated Protein C Resistance (APC-R)
FVa is usually inactivated by APC-Protein S complex , in FVL is there is a mutation on FV molecule
Maintains normal co factor ability so coagulation is normal
arginine is the site of APC/PS cleavage
Va not inactivated!!! > Remains active
No reduction in prothrombinase production
Continued clotting
F V Leiden / APC-R Lab Analysis
-suspected based on history
PT and PTT is normal
screen with APC-R assay
pt plasma+ FV deficient plasma (all factors but V )
that way you are assessing only the presence of Fv in pt plasma
Two aliquots
both contain APTT reagent + FV-depleted normal plasma + Patient plasma
First aliquot:
1) Add CaCl
2)Measure time to clot
Second aliquot
1)Add CaCl and APC.
2)Measure time to clot
APC should inactivate patient’s FVa and prolong APTT
–Second aliquot should be prolonged by AT LEAST 1.8x of the first aliquot
If person is APC-Resistant (aka has FVL) > there would be normal clotting bc FV is not inactivated (Fv L mutation confirmed by molecular genetics)
determine zygosity as homozygous state is high risk
LAC interferes with APC-R so go directly to genetics
Prothrombin G20210A
prothrombin mutation FII
G to A mutation
increased risk of DVT
increased Prothrombin level = more thrombin
-molecular genetics to diagnose
Dysfibrinogenemia
-functional or structural abnormality of the fibrinogen molecule
-Abnormalities in structure results in resistance to plasmin
-Plasmin cant recognize cleaving site so the clot won’t be able to be digested
-This means clotting will continue
Test - Fibrinogen concentration or Thrombin Time (TT) Assay
Decreased Fibrinogen levels
Abnormal TT (prolonged) bc abnormal structure makes it so there’s issues with cleaving fibrinogen to fibrin
Plasminogen:
how does the assay work
Without plasminogen, patient is unable to breakdown clots
Use of thrombolytic therapy (e.g Recombinant TPA) useless without plasminogen
Chromogenic Substrate Assay can be used to measure Plasminogen levels
1)Patient’s plasma is mixed with Reagent containing streptokinase (plasminogen activator)
2)Patient’s plasminogen will be converted to plasmin
3)Plasmin will react with chromogenic substrate to release colour
Colour intensity will be proportional to the concentration of plasminogen in patient
Tissue Plasminogen Activator
how does the assay work
deficiency associated with Myocardial Infarction (MI), stroke & DVT
-Without TPA, plasminogen cannot be converted - no clot breakdown
Chromogenic Substrate Assay to measure TPA activity:
1)Known constant conc of Plasminogen is added to patient plasma
2)Patient’s TPA will convert plasminogen to plasmin
3)Plasmin will react with chromogenic substrate
Intensity of colour substrate is proportional to TPA activity
Plasminogen Activator Inhibitor 1
Deficiency associated with mild bleeding
INCREASED PAI-1 = thrombosis, primary inhibitor of fibrinolysis – increased levels stops TPA from activating plasminogen – clots don’t get broken down
PAI-1 overproduction is measured using indirect approach:
1)Patient plasma is mixed with a fixed amount of reagent and TPA in excess
2)Patient’s PAI-1 will inactivate reagent TPA.
3)The residual TPA that hasn’t been inactivated will activate reagent plasminogen to plasmin. This will react with chromogenic substrate
Colour intensity is INVERSELY proportional to patient’s PAI-1 activity
Factor XII, PK, HMWK deficiency
involved in activating plasminogen , if there is a deficiency then fibrinolytic activity is reduced
FXII deficiency doesn’t always result in bleeding disorders it can be bypassed as a contact factor by direct activation of FXI thrombin
Tissue Factor Pathway Inhibitor (TFPI) Deficiency
inhibits the Extrinsic Tenase complex
TFPI binds directly and inhibits the Xa & TF–FVIIa complex therefore inhibiting continued activation of FXa
Low levels of TFPI are a risk factor for clotting disorders
Allows for continued FXa activity
