Coagulation studies Flashcards
Steps in investigations of abnormal coagulation profile
- Rule out pre analytical, analytical and post analytical issues
- If only PT/INR and APTT are prolonged check for warfarin therapy.
- Fibrinogen and D-dimer levels can exclude DIC.
- TCT will exclude heparin, dabigatran or paraprotein.
- An anti-Xa assay is useful to exclude LMWH, UFH, rivaroxaban or apixaban.
- Mixing test may or may not be useful here as some inhibitors a time dependant.
- If all these are normal, running specific clotting factor assay at a wide range of dilutions are useful.
Bethesda assay principle
- Inhibitors are antibodies that form against specific clotting factors (e.g. f8, f9)
- They may be time-dependent (e.g. f8) or immediate acting (e.g. f9)
- They can develop in patients with haemophilia (allogenic), or without a bleeding disorder (autoantibodies)
- Assay to quantify amount of a factor VIII inhibitor present.
- Serial dilutions of the patient sample are incubated for 2hrs at 37ºC with a known amount of factor VIII.
- Amount of FVIII in the incubation mixture is corrected for the expected deterioration of FVIII after 2hrs by comparing:
o FVIII in the normal plasma + patient plasma mixture
o to that of a mixture of normal plasma and diluent (referred to as the control tube). - 1 Bethesda Unit corresponds to 50% residual Factor VIII after 2hrs incubation at 37°C
o Aka the amount of inhibitor required to inactivate half of the Factor VIII present in the patient plasma/NPP test mixture. - Results are expressed as Bethesda units/mL (normal range 0 – 0.5 BU/mL)
Sample requirements for bethesda Assay
- PPP
- Patient plasma heated to 58°C for 90mins then centrifuged to remove precipitated proteins.
o In patients receiving treatment with a factor VIII concentrate, the measurement of inhibitors can be problematic as residual factor VIII may be present
o Heat treatment/inactivated PPP to a FVIII:C and FVIII:Ag of <0.10 IU/ml.
o Igs are heat resistant so the inhibitor level in the plasma sample is unaffected. - This resulting supernatant is free of all clotting factors, but still contains the IgG inhibitor in an active form.
Bethesda Assay Method
- Control Tube 1: Equal parts of commercial FVIII deficient plasma is mixed NPP (buffered to pH7.4) .
- Patient Tube 2: Equal parts of heat inactivated PPP is mixed with NPP
- Serial dilutions are made of the heat inactivated PPP with the commercial FVIII deficient plasma to titrate the antibody.
- All tubes are covered and incubated at 37 degrees for 2 hrs. Time to allow the inhibitor to fully express itself.
The use of the control corrects for the deterioration in f8 and f5 during the incubation period.) - A factor 8 assay is now performed on all tubes using a standard one stage factor VIII assay
- The percent residual FVIII per patient tube is calculated using the following equation:
- “Residual factor activity is defined as the relative percentage factor activity of the test mixture compared to the control mixture”
- Only residual factor VIII% between 25% and 70% should be taken from the graph.
- A log-linear plot reflecting the correlation between residual activity FVIII% and inhibitor level is used to determine the patient inhibitor level
- The dilution factor for each tube is applied to the graph reading to obtain BU/ml which is then multiplied by the dilution factor
- residual FVIII levels between 25% and 70% should derive comparable inhibitor levels from the graph once corrected for dilution.
- Where increasing dilutions do not give acceptable duplicates the antibody is displaying non linearity. In this instance the dilution closest to residual factor VIII of 50% has been found to be the most clinically useful.
(The incubation step is not required for a f9 inhibitor, since these are immediate acting.)
Factor VIII inhibitor detection by ELISA
- Recombinant f8 is immobilized on a microtitre plate, and diluted patient plasma is added to wells and incubated
- If an anti-f8 antibody is present, it will bind to the immobilized f8
- The plate is washed, and any unbound material is removed
- Labelled anti-IgG is added to the wells and incubated
- The unbound anti-IgG is removed by washing, and substrate (which reacts with the label of the IgG) is added that can be measured at an optical density at 405 nm, from which the presence of a f8 antibody can be established
- This method picks up both neutralizing and non-neutralizing antibodies, unlike the Bethesda, which only picks up neutralizing antibodies
APTT (Activated Partial Thromboplastin) summary
- Clot-based test that evaluates the overall efficiency of the classical intrinsic pathway – APTT reagent = Actin FS (purified
soy phosphatides) - Sample: platelet-poor plasma (PPP) – citrate tube
- The APTT measures the clotting time of plasma after the addition of a contact activator (reagent used is Actin FS), phospholipid and CaCl2 – but without tissue thromboplastin
- It depends on the contact factors within the classical intrinsic pathway (8,9,10, 12), as well as X, V, prothrombin and fibrinogen
- It is also sensitive to the presence of circulating anticoagulants (inhibitors) and heparin
APTT/PT/Fibrinogen and TCT QC
Internal QC:
- commercial normal and abnormal controls, tested with every new vial, at 7am (startup) and then 8 hourly
All other tests must have controls run with each batch, change of reagent vials or at least every 24 hours. EG: DDimer, Lupus, APCR, VWF
External QC:
- RCPA QAP (2 samples, 4/year)
Warfarin MOA
Warfarin competitively inhibits the vitamin K epoxide reductase complex subunit 1 (VKORC1).
–> blocks the gamma-carboxylation of glutamic acid residues of the Vitamin K−dependent coagulation factors II, VII, IX and X.
Heparin MOA
UFH is a polysaccharide with MW of 3-30 kDA.
Works indirectly by binding to AT –> causing a conformational change –> converts AT from a slow to a rapid inactivator of coagulation factors.
LMWH is purified from UFH has a more uniform size between 3.5 – 5 kDA
UFH is able to accelerate (up to 2000 fold) the inhibitory effect of antithrombin,
Heparin AT complex inactivate factors IIa (thrombin), Xa, IX, XI, XII and plasmin.
UFH, LMW hepаrin, and fondaparinux all inactivate factor Xa, but unfractionated heраrin also inhibits thrombin (due to the long heparin chain)
Fondaparinux appears to have nearly pure anti-factor Xa activity.
Heparin Resistance
Requirement of unusually large doses of heparin in order to achieve APTT in therapeutic range.
- eg > 35 000 units of heparin in 24 hours, excluding initial bolus and infusion rate of >400 units/hour in CAGS patients
Some predictors of heparin resistance:
- baseline AT activity level <60%,
- platelet count >300,
- age >;65
Causes of Heparin resistance
- Antithrombin deficiency
> sepsis, liver disease, ECMO, bypass, congenital - Heparin-binding plasma proteins ( factor 8, vWF, acute phase reactants)
- Increased heparin clearance
Laboratory investigation of heparin resistance
Ensure correct collection of sample. Not from heparin line/arm
Recommendation is Anti-Xa as excludes interaction from upstream effects
APTT
–> PPP, Actin FS (contact factor activator), calcium and phospholipid.
–> measured by either optical of mechanical endpoints
–> usually first test to identify heparin resistance
Anti-Xa
–> Citrated patient plasma is added to an excess of factor Xa;
heparin and AT in the patient plasma will inhibits factor Xa;
residual Xa activity is then measured via cleavage of a chromogenic substrate and is inversely proportional to the anticoagulant present
UFH target is 0.3-0.7
LMWH target is 0.5-1.0
Advantages over APTT include being insensitive to low factor levels (e.g. in DIC), high factor 8 levels (e.g. in critical illness), or antiphospholipid antibodies
Chromogenic assays are affected by haemolysis, bilirubin and lipids
ATIII levels
–> Plasma is incubated with heparin and excess factor 2a
–>ATIII in the patient plasma complexes with the heparin/factor 2a, inhibiting factor 2a
Residual factor 2a cleaves a chromogenic substrate, which is measured optically at 405nm, and is inversely proportional to the ATIII concentration in the sample
Alternative reagents include bovine factor 2a; or bovine factor 10a
Will overestimate ATIII levels in the presence of an anti-Xa DOAC if the 10a method is used (and in the presence of dabigatran if the 2a method is used)
Management of heparin resistance
little evidence for antithrombin replacement in acquired antithrombin deficiency
Recommendation to continue uptitrating heparin to therapeutic antiXa levels (0.3 - 0.7)
