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Flashcards in Coagulation Studies Deck (47):

Reagents in APTT test

Patient poor plasma (PPP)
Kaolin/Silica/Gallic acid derivative (activator)
Cephalin (phospholipid)
==> incubate for 4 mins then add calcium and time until clot formation occurs.


Describe the prothrombin time

PPP is mixed with thromboplastin (TF and phospholipid).
Calicum is added to initiate clotting and the time to clot formation is recorded.


What is an ACT (activated clotting time)

- POCT used predominantly to monitor heparin therapy during cardiac surgery and ECMO
- Utilises whole blood + activator e.g. Kaolin
- No need to add phospholipid as there are patient platelets present
- Time to clot formation is recorded
- The ACT is not specific for heparin and can be prolonged by hypothermia, platelet dysfunction etc.


How do FDPs cause a prolonged TCT?

They interfere with fibrin polymerisation


Protamine sulfate neutralises the effects of (2)

1. Heparin
2. FDPs


Name 5 different ways of measuring fibrinogen level

1. Clauss (functional) = recommended
2. PT based (not recommended)
3. Immunological (antigen not function)
4. Gravimetric eg. fibrin clot weight
5. TEG


What are two screening tests for dysfibrinogenaemia?

1. TCT
2. Reptilase time (not affected by DTIs, heparin) - prolonged with low/abnormal fibrinogen and FDPs


How is dysfibrinogenaemia diagnosed?

1. Screening tests - TCT and Reptiliase time
2. Check fibrinogen activity/antigen ratio
==> Clauss fibrinogen method and immunological method
3. LFTs
4. Fibrinogen electrophoresis
5. Molecular and family studies


What are PIVKAs?

Proteins Induced by Vitamin K Antagonism
==> Factors with glutamic acid residues that haven't been gamma carboxylated therefore the coagulation factors are biologically inactive.


How does Vitamin K work?

Causes gamma carboxylation of glutamic acid on factors II, VII, IX and X


How does warfarin work?

It inhibits vitamin K epoxide reductase and therefore vitamin K is not in a form to allow gamma carboxylation of coagulation factors


What does the INR correct for?

The INR corrects for the different thromboplastin sensitivities to reductions in Vit K dependent coagulation factors compared to the WHO reference standard.


Why is a local calibration system for thromboplastins advised?

The ISI of thromboplastins can vary depending on the machine and endpoints.


What methods are recommended to measure dabigatran concentration in plasma? (4)

Gold standard = mass spectometry
Specialised tests which have a linear dose-response curve:
- TCT using dilute patient plasma OR
- Ecarin clotting time/chromogenic assay (independent of fibrinogen concentration)
- Anti-IIa chromogenic assay
*PiCT not advised
*Global assessments of coagulation such as TEG, ROTEM, ACT and TGA not recommended


Name three methods for measuring rivaroxaban levels

1. Mass spectometry
2. Anti-Xa assay
3. dRVVT
*PiCT not advised
*Global assessments of coagulation such as TEG, ROTEM, ACT and TGA not recommended


Tests for detecting factor XIII deficiency

Normal APTT and PT
Screen = clot solubility test (allow plasma to clot, put sample in urea for 24hrs, if it dissolves then suspect severe deficiency)
Immunological - ELISA for A and then B subunits
Functional assay - based on the transglutaminase activity of Factor XIIIa. NH3 released from glycine ethyl ester, NH3 reacts with NADPH to form NADH (decreased fluorescence) and glutamate.


Describe the AT assay in your lab

Functional assay based on the heparin cofactor activity of antithrombin.
Bovine thrombin and heparin are added to patient plasma. Residual thrombin (IIa) is measured using a chromogenic assay (pNA). Colour change is inversely proportional to AT activity.
Interference - DTIs, jaundice, lipaemia, haemolysis
Heparin in sample does not interfere.
Bovine thrombin is chosen as heparin cofactor II does not interfere with this.


Name 3 different solutions for EDTA mediated platelet clumping

1. Alternative counting method from impedance eg platelet-O channel (optical with DNA and RNA stains to identify WBCs and retics and platelets; tested at 41 degrees so no interference by cryoglobulins)
2. Adding aminoglycoside - dissociates clumps)
3. Alternative anticoagulant
- citrate
- MgS04 (thromboexact)


Different methods for measuring antithrombin levels

1. Functional - based on AT's activity against factor II and factor X (chromogenic assays). We use bovine thrombin as it is not sensitive to heparin cofactor II effect
2. Immunological - latex particle immunoassay


How would you investigate possible antithrombin deficiency?

1. Clinical and family history ?thrombosis
2. Measure antithrombin activity using functional chromogenic IIa assay
3. Measure AT using immunological method (compare antigen/function ratio)
4. Molecular - sequence antithrombin gene (SERPINC1) gene.


What different AT mutations are there?

1. Quantitative
2. Qualitative - mutations can affect the heparin binding site, reactive site or both.


How does your lab test for protein C deficiency?

Chromogenic assay - protein C in patient plasma + Protac ==> APC acts on chromogenic substate to for pNA.
- not affected by protein S
- not affected by FVL mutation or high levels of factor VIII


Causes of low protein C?

1. Inherited (quantitative or qualitative defects - quantitative defects are more common, usually point mutations)
2. Acquired:
- acute thrombosis
- liver disease
- nephrotic syndrome
- vitamin K deficiency and warfarin


Name some problems associated with a clot-based assay for protein C

1. Activated protein C resistance - falsely low levels of PC
2. Anything that prolongs baseline APTT e.g. factor deficiency, LA, LMWH will cause falsely high levels of PC


Describe the APTT-based protein S assay in your lab?

1. Patient plasma is mixed with PS-deficient plasma
2. Factor Va, activated protein C and phospholipid are added.
3. After incubation calcium is added and the time taken for clot formation is measured (clotting is activated at the common pathway)
4. If the lines are linear, can measure protein S from standard curve.
**addition of PS deficient plasma and factor Va reduces interference from APC resistance.**


What is 60% of protein S bound to?

C4b-binding protein


Causes of low protein S

1. Inherited
2. Acquired
- pregnancy, OCP, HRT
- liver disease
- warfarin, vitamin K deficiency
- nephrotic syndrome
- acute thrombosis
- Sickle cell


Describe the mechanism of APC resistance with the FVL mutation

FVL = mutation in factor V which makes it resistant to APC degradation (cleavage site changed by mutation)


Describe the test for APC resistance

APTT is run on patient plasma diluted in FV-deficient plasma with and without added APC. Ratio <2.0 = APC resistance.
i.e. plasma does not show significant prolongation in clotting when APC is added.


How to distinguish liver disease from DIC?

1. Clinical judgement
2. Factor VIII usually low in DIC but normal in liver disease (produced by endothelial cells)
3. D-dimer higher in DIC
**significant overlap, can get increased fibrinolysis in liver disease. APTT and PT prolonged, fibrinogen low in both.


How do you distinguish vitamin K deficiency from liver disease?

1. Echis ratio - prolonged in liver disease, normal in vit K deficiency
2. Check factor levels - FV will be normal, FVII will be low.


How would you manage bleeding in a patient with liver disease?

1. Assess the cause of bleeding ==> variceal bleeding is treated differently from non-variceal bleeding
2. Assess coagulation
- APTT, PT, fibrinogen
- Platelet count
- Is the patient on anticoagulation
3. Optimise coagulation status
- Vitamin K, FFP, platelets, cryo
4. Tranexamic acid to inhibit fibrinolysis
5. Variceal bleeding is due to increased portal pressures - manage by reducing portal pressure (TIPS) and variceal ligation.


Management of thrombosis in a patient with advanced liver disease?

1. If the patient has varices then they are at high risk of bleeding and may not be suited for anticoagulation
2. Anticoagulation - difficult if baseline INR is raised to use warfarin, consider DOAC, LMWH


What is the mechanism of acquired dysfibrinogenaemia in liver disease?

Increased sialic acid residues increase the net negative charge and impair fibrin polymerisation.


Differential diagnosis of prolonged PR but normal echis ratio?

1. Vitamin K deficiency
2. Warfarin
3. Factor VII deficiency (echis turns prothrombin into meizothrombin ==> cascade is activated below FVII)


New agents in haemophilia

Gene therapy using viral vectors
Extended half life products (protein modification, pegylation, fusion with Fc portion of IgG or albumin)
Alternate methods of activation = emicizumab bispecific antibody that bridges factors IX and X
Rebalancing haemostasis
1. concizumab = antibody against TFPI
2. fitusiran = inhibitory RNA against AT
3. antibody against activated protein C


Pathogenesis of the antibody in acquired haemophilia

1. Binds to FVIII and clear it
2. Binds to VWF binding site and disrupts binding
3. Binds to FIX or FX binding sites and interferes with the tenase complex


Gamma carboxylation of glutamic acid residues on Vitamin K dependent factors allows what to bind?

Calcium (and therefore attachment to phospholipids)


How is combined deficiency of vitamin K dependent factors treated?

1. High dose vitamin K (usually only mild cases)
2. FFP


Benefits of TEG and ROTEM over laboratory coag tests (3)

1. Fast and POCT
2. Provide an assessment of whole blood coagulation (may be more similar to in vivo)
3. Provide an assessment of fibrinolysis


Limitations of TEG and ROTEM (3)

1. Can't reliably assess the effects of anticoagulants (warfarin, DOACs)
2. Not sensitive to platelet disorders, anti-platelet agents or vWD
3. High or low haemoglobin can affect the results


If a contact factor deficiency is suspected, what can be done with the APTT to prove this?

Compare a short and long incubation time - short incubation time = sensitive to contact factor deficiency, long incubation time is insensitive.


What are two advantages of photo-optical end-point detection?

1. PT-derived fibrinogen
2. Biphasic waveform seen in DIC


How does protamine work to inhibit heparin?

Heparin is highly negatively charged and protamine is highly positively charged.


What are the causes of a prolonged TCT?

1. Hypo/dysfibrinogenaemia (inherited and acquired = liver disease, DIC, thrombolysis, PLEX)
2. Anticoagulants - UFH, DTIs
3. FDPs (interfere with polymerisation)
4. Paraprotein (interferes with polymerisation
5. Low albumin


Name causes of a prolonged reptilase time

Reptilase converts fibrinogen to fibrin.
Prolonged reptilase time is caused by low fibrinogen and dysfibrinogenaemia and FDPs.


Describe the effect of 1. Heparin and 2. DTIs on the echis ratio and reptilase time.

Echis: prolonged by DTIs but not heparin.
Reptilase: not affected by DTIs or heparin.