Past Coag VIVA Qs Flashcards
Samples for coag
9:1 sodium citrate anticoagulated blood for coag
Citrate adjusted bottles for very high/very low Hct
Routine tests
PT
APTT
Thrombin Time/Fibrinogen
D-Dimers
INR
Analyser
ACL TOP 550
How is PT calculated
§ PT is worked out using the first derivative curve of the clot curve
§ In particular the peak of the first derivative curve which coincides with the maximum speed of the clotting reaction is equal to the time it takes for the clot to form
Peak of first derivative curve = PT time in seconds
How is APTT calculated
§ Analyser gets the end-point of fibrin clot formation and then finds the 2nd derivative curve for this
§ The second derivative curve peak coincides with the max change in velocity of the curve/speed of clot formation
The point of fastest clot formation is taken as the APTT clotting point of the sample
How is fibrinogen/thrombin time calculates
§ Clauss method of measuring fibrinogen
§ Fibrinogen is quantified using the thrombin time curve
□ Based on the time taken for thrombin to convert fibrinogen to fibrin (Thrombin time)
§ The thrombin time is directly proportional to the amount of fibrinogen
□ Higher fibrinogen levels = shorter clotting times
A calibration curve is used to calculate the fibrinogen concentration
How are D-dimers calculated
§ Measured turbidometrically
§ Patient plasma is mixed with D-dimer speicific antibodies which bring about agglutination
The degree of agglutination is proportional to the amount of D-Dimer present in the patient sample and read from a stored reference curve
Controls
○ Daily QC -> multi-level controls
Inter-analyser controls
EQA
What to do with prolonged PT
○ Check for clot
○ If clotted then ask for repeated sample
○ If no clot:
□ Check patient history
○ If this is the first time it is low then investigate i.e. repeat
○ If previously prolonged -don’t repeat
○ Check if on warfarin?
○ Look at liver function tests
○ Mixing studies
Factor assays for extrinsic pathway
What to do with prolonged APTT
○ Check for clot
○ If clotted then ask for repeated sample
○ If no clot then phone result
○ Look at liver function tests
○ Check if on heparin?
○ Mixing studies
○ Lupus anticoagulant screen
Factor assays for intrinsic pathway
What tests make up the thrombophilia screen
Protein C, Protein S, Antithrombin, Factor V Leiden, Lupus anticoagulant
For who would we do a thrombophilia screen and why
○ Done for patients experiencing thrombophilia i.e. increased blood clots -> deep vein thrombosis etc
We are investigating for deficiencies of naturally occuring anticoagulants
What is protein C
§ Vitamin K dependent protein
§ Anticoagulant
§ Activated by a serine protease into activated protein C (APC)
APC inactivates factors V and VIII preventing coagulation
How is protein C measured
□ Chromogenic assay
□ Protein C is activated using venom of Copperhead snake
□ Protein C activator + chromogenic substrate incubated with patient plasma
□ As protein C is activated to APC a chromogen is produced
The signal is directly proportional to the amount of activated protein C
What is protein S?
§ Vitamin K dependent protein
§ Anticoagulant
§ 2 forms: free protein S (40%) and protein S linked to complement C4 binding protein (C4BP) (60%)
§ Only free form = Co-factor of activated protein C
□ Potentiates the anticoagulant function of protein C
Inactivates factor V and VIII
How is protein S measured
□ Turbidometric
□ Measure free protein S
□ Use C4 binding protein buffer + latex + anti protein S monoclonal antibody latex
□ Any free protein S present in patient sample will be bound up by C4
□ The specific monoclonal antibody will then bind to this complex to form agglutination
□ Amount of agglutination measured turbidometrically
Degree of turbidimetry directly proportional to amount of Protein S
What is antithrombin
§ Heparin cofactor I
□ Heightens the affect of heparin
□ Essential for effective heparin therapy
§ Prevents uncontrolled coagulation/thrombosis
Inhibits coagulation proteases especially thrombin, Factor Xa and Ixa
How is antithrombin measured
□ Indirect measurement of antithrombin by measuring the amount of Xa in plasma
® High Xa in plasma = low antithrombin
□ Liquid Xa reagent + chromogenic substrate used
□ Heparin + a known amount of FactorXa are incubated with patient plasma
□ Patient plasma contains some antithrombin
□ Any antithrombin present in patient plasma will react with the FXa
□ Any Fxa left over reacts with the chromogenic substrate to produce a measurable chromogen
The more Fxa left over/the higher the FXa signal the lower the amount of antithrombin in the patient sample
What is factor V leiden/Activated protein C factor V assay
An indirect way of screening for the presence of FVL mutation
How do we carry out the APCFV assay
□ Activated protein C is added and the clotting time measures
□ If FVL mutation is present it will prevent the activated protein C from acting on it -> thus coagulation will occur quickly
□ If normal FV the APC will act on it to inhibit coagulation
Any short APCFVs are sent to St. James’ for genotyping of the FV mutation
What is lupus anticoagulant
Autoimmune phospholipid antibodies which interfere/prolong phospholipid dependent coag tests such as APTT
What are the two assays for Lupus anticoagulant
dRVVT (screen + confirm)
Silica clotting time (screen + confirm)
What is dRVVT
Dilute Russel Viper Venom (dRVVT) is mixed with platelet poor plasma
-> should cause clotting if phospholipid is present
-> won’t form clot is Lupus anticoagulant has removed phospholipid
How do we carry out the dRVTT assay
◊ Dilute Russel Viper Venom (dRVVT) is mixed with platelet poor plasma
◊ dRVVT activates FX -> FXa (should cause coag if phospholipid present)
} Screen
– Poor in phospholipid i.e. sensitive to LA
} Confirm
– Contains additional phospholipid
– If patient has LA the confirm will neutralise anti-phospholipid antibodies i.e. it should shorten clotting time
How do we carry out the SCT assay for LA
◊ Platelet poor plasma is mixed with silica particles (activator) which initials coag cascade. If phospholipid present/no LA then coag should occur)
◊ Screen
} Poor in phospholipid
} Will be prolonged if LA
◊ Confirm
} High in phospholipid
Will correct if LA
Talk about FVL mutation
§ Most common Factor V mutation -> out of all mutations it confers the lowest risk of clotting
§ FVLM prevents activated protein C from working on the factor V
Activated protein C inhibits coagulation by inactivating factor V (as well as factor VIII)
What is our warfarin clinic for
○ Monitoring of warfarin treatment
○ Require INR calculated weekly
Moving most patients over to DOACs now
How to calculate INR
[Patient’s PT/Mean Normal PT] (ISI)
How is MNPT calculated
Geometric mean of 20 normal patients
What is ISI
○ ISI = International Sensitivity Index = the sensitivity of the thromboplastin reagent when compared with the WHO Reference Thromboplastin where MNPT:The geometric mean of 20 normal patients
What is the INR reference range
○ Normal INR = 2
○ INR for someone with anti-phospholipid antibodies (LA) = 3
INR >5 is very high and must be phoned to clinical -> risk of clotting
What would you do with an INR >5
○ Check for clot
○ If no clot then repeat test
○ If clotted then ask for repeat sample
○ If true result
Phone result to clinican
Why do we need to calculate INR
○ To monitor warfarin treatment
○ Must be kept in therapeutic range
Enough warfarin to stop clots but not too much that patient is at risk of clot
What might increase D-dimers
disseminated intravascular coagulation (DIC)
deep vein thrombosis (DVT), pulmonary embolism (PE),
Surgery/Trauma
Pregnancy
What factors are involved in the common pathway of coag?
Common = I, II, V, X, XII
What factors are involved in the extrinsic pathway
Common = I, II, V, X, XII
Extrinsic = III, VII
What factors are involved in the intrinsic pathway
Common = I, II, V, X, XII
Intrinsic = VIII, IX, XI, XII
What is factor III also called?
Tissue factor
What is the principle of the ACL TOP 550?
- The analyser produces a clot signature curve for every reaction
- This curve is a visual representaation of the coagulometric or absorbance reaction taking place
The time taken for a fibrin clot to form which is determined using the reduction in the amount of light transmitted through the sample as the end-point
- This curve is a visual representaation of the coagulometric or absorbance reaction taking place
List the DOACs
Dabigatran
Rivaroxaban
Apixaban
Argatroban
How does apixaban work and how is it measured?
Directly inhibits factor Xa
Measured using an anti-Xa assay
Inversely proportional to Apixaban concentration
How does rivaroxaban work and how is it measured
Directly inhibits factor Xa
Measured using an anti-Xa assay
Inversely proportional to rivaroxaban concentration
How does dabigatran work and how is it measured
Direct thrombin inhibitor
Measured using thrombin time? -> addition of thrombin to patient plasma -> time taken for fibrin clot to form -> longer the time the more dabigatran etc
TT is directly proportional to dabigatran concentration
How does argatroban work and how is it measured
Direct thrombin inhibitor
Measured using thrombin time? -> addition of thrombin to patient plasma -> time taken for fibrin clot to form -> longer the time the more argatroban etc
TT is directly proportional to dabigatran concentration
How does warfarin work?
Warfarin blocks on of the essential enzymes that uses vitamin K to produce clotting factors
Depletes functional vitamin K reserves to thereby reduce the synthesis of active clotting factors
What would you do if QC failed?
- Check has both QC failed or just 1
- If just 1 QC has failed then only a single reagent has failed- Make up new QC
- If it fails again
- Make up new reagent
- Ring engineer
- Recalibrate
Will have to backtrack through results -> retest and see how many results will need to be ammended
- Make up new QC
Describe the treatment of a lupus patient
- Haematology measures the amount of lupus anticoagulant while immunology measures the lupus antibodies to evaluate risk
- A patient is at x3 times the risk if they have anti-phospholipid antibodies
- These patients are not suitable for DOACS
They will need a higher dose e.g. warfarin etc
What are the vitamin K dependent clotting factors?
II, VII, IX, and X
Mode of action of heparin
Catalyses the inactivation of thrombin by Antithrombin III (ATIII)
