Coagulation Tests Flashcards

1
Q

List 5 different roles that thrombin plays in normal coagulation

A

1) Amplification of initial thrombosis formation:
- Activates FV, FVIII and XI to enable formation of the tenase and prothrombinase.

2) Activation of platelets
3) Formation of fibrin from fibrinogen

4) Binds to thrombomodulin:
- Leads to an alteration in thrombin substrate specificity: proteolyses PC to APC which, along with its cofactor PS, inactivates FVa and FVIIIa.

5) Causes the release of TAFI from platelets:
Inactivates TF-FVIIa complex.

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2
Q

What are the three different types of ways to measure fibrin formation (end point detection) in coagulation assays?

A

1) Optical method: Optical changes occur as fibrin forms (clear to opaque colour change)
2) Electromechanical: Viscosity based. Fibrin strands prevent ball movement
3) Impedance

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3
Q

How is a PT performed and in what 6 situations is a PT used

A

1x vol of pre-warmed plasma + 2x vol of pre-warmed thromboplastin + calcium. Time to clot formation measured.

1) Factor deficiency- inherited or acquired
2) Conversion to INR for warfarin monitoring
3) Adapted for factor assays (II, V, VII, X)
4) Adapted to direct Xa inhibitor assays
5) Adapted for LA testing
6) Prognostic marker in liver disease

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4
Q

Discuss how the INR is calculated

A

Different thromboplastins have different sensitivities to the defects in coagulation induced by warfarin.
INR allows uniformity in warfarin dosing between labs.

INR= Patient PT/ MNPT raised to the power of the ISI

MNPT: Geometric mean PT of at least 30 normal plasmas

ISI: International sensitivity index. Enables the sensitivity of a particular thromboplastin to warfarin to be calibrated against an International WHO reference thromboplastin.

(PR= PT/MNPT)

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5
Q

How is an APTT performed and what are the 6 common uses for APTT?

A

1x vol plasma plus 1x vol APTT reagent (phospholipids and activator). Incubated for 3-5 minutes. Calcium added. Time to clot formation measured.

1) Factor deficiency (acquired or inherited)
2) Lupus anticoagulant detection
3) Heparin monitoring

4) Adapted for factor assays (VIII, IX, XI, XII, prekallikrein)
5) Adapted for LA testing
6) Adapted for APC resistance testing

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6
Q

What five variable influence APTT results?

A

1) Phospholipid used (affects responses to coagulation defects or inhibitors of coagulation.)
2) Activator used
3) Buffers used
4) incubation time
5) Clot detection method used

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7
Q

What constitutes a correcting APTT?

A

Varied definitions:

1) Rosner index (ICA): (Mix - Normal) / Patient x100
- Typical cut-off for factor deficiency= ~<12%
2) Percent correction: (Patient-mix)/ (patient- normal) x100
3) Percent of mix above normal: (Mix - normal)/ normal x100
4) Normal + 5 seconds
5) Is mix within the reference range?

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8
Q

Discuss thrombin time

A

Neat test plasma + 2NIH units/ml of thrombin. Neat clot time recorded in sec. No calibration curve.

Use of neat plasma and low thrombin concentration makes the test sensitive to prolongation by thrombin inhibitors

Thrombin time is also prolonged in: hypo, dys or afibrinogenaemia, myeloma paraproteins, high FDPs

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9
Q

Discuss the Clauss fibrinogen method

A

Plasma diluted 1/20 in a buffer + 100NIH units/ml of thrombin. Calibration curve formed using a WHO calibration plasma. Graph converts CT to result in g/L.

Use of highly diluted plasma and high thrombin concentration makes the test insensitive to thrombin inhibitors.

The clotting time is therefore responsive only to fibrinogen concentration of the test plasma

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10
Q

How is the chromogenic anti-Xa assay performed?

A

1) Patient plasma at 1 in 2 dilution
2) Chromogenic substrate
3) Bovine factor Xa added

The patients plasma will contain heparin attached to anti-thrombin. This complex inhibits the Xa in the reagent leaving a residual amount of Xa. The residual Xa is given a substrate which causes a colour change.
Rate of colour formation 1/α to heparin concentration.
The heparin concentration is then read off a calibration curve.

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11
Q

How is the therapeutic APTT range for heparin determined?

A

By correlating APTT results with anti-Xa results

  • Using ex-vivo samples, simultaneously measure the APTT and anti-Xa level.
  • Regression line of best fit drawn.
  • Line drawn on either side of the therapeutic range of heparin (anti-Xa level of 0.3 and 0.7).
  • The therapeutic range is the area between where these lines intersect with the APTT on the X axis
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12
Q

What is the protamine sulphate neutralisation test?

A

Test plasma incubated with buffer at 37deg and thrombin added.
If the sample clots within 10s- no UFH is present and no further tests are necessary.
If the TT is prolonged, different dilutions/ concentrations of protamine sulphate are used until a concentration is found that corrects the TT. This can be used to determine the concentration of heparin in a sample.
Therapeutic range of heparin can be established by determining the aPTT range that corresponds to 0.2 to 0.4 units/mL by protamine titration.

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13
Q

What is the reptilase test

A

Can be performed to determined why the TT is prolonged

  • Snake venom, binds fibrinogen at a different site from thrombin, converting it to fibrin
  • Test is essentially the same as the TT although reptilase is added instead of thrombin.

○ Prolonged reptilase time is seen in:

  • Inherited and acquired hypofibrinogenaemia/ dysfibfinogenaemia
  • High levels of FDPs
  • Hypoalbuminaemia (in vitro phenomenon that also prolongs the TT)
  • Liver disease (mutlifactorial: hypo and dysfibrinogenaemia, elevated FDPs)
  • Neonate
  • Myeloma (some paraproteins interfere with fibrin polymerisation and lead to a prolonged TT and RT).

• Prolonged TT, normal RT= UFH, direct thrombin inhibitors

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14
Q

How is the one stage factor assay performed?

A

1) Standard curve made
- APTT/PR performed using serial dilutions of a reference plasma mixed with an equal volume of factor deficient plasma

2) Patients sample tested
- APTT/ PR performed on at least 3 serial dilutions of patients plasma mixed with an equal volume of factor deficient plasma ( In the presence of other APTT substrates)

3) Patients factor levels are read off/ calculated from the standard curve

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15
Q

How is the two stage chromogenic factor assay performed?

A
  • Reagent cocktail for generating Xa (everything except FVIII) mixed with a chromogenic substrate for Xa and the patients plasma.
  • The Xa produced will be cleaved by the chromogenic substrate and absorbance measured by a spectrophotomer
  • Results read off a standard curve
  • As FVIII is the rate limiting step in Xa production, the amount of absorbance produced is directly proportional to the amount of FVIII in the sample.
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16
Q

What is the definition of a non-linear result on a factor assay?

A

If the factor result at each dilution differs by >10%, then the assay is said to be “non-linear”.

17
Q

What are the Nijmegen modifications of the Bethesda assay?

A

Nijmegen modifications applied to most modern Bethesda assays to increase sensitivity, specificity and accuracy. These modifications include:

○ Using plasma that is deficient in the factor that is suspected to be affected by the inhibitor.
○ The normal pooled plasma is buffered to a pH of 7.4

18
Q

What are the steps of the Bethesda assay?

A

1) Heat patient plasma to 58deg for 90 minutes to destroy patient clotting factors but retain the IgG inhibitor
2) Set up the control tube containing normal pooled plasma and FVIII deficient plasma (i.e. no inhibitor present)
3) Make serial dilutions of the test patients plasma. Equal volumes of FVIII deficient plasma and normal pooled plasma added to each tube. An equal amount of patient plasma added to the first tube then a serial dilution is made.
4) Tubes covered and incubated at 37deg for 2hrs.
5) FVIII assay run

6) Record the residual FVIII% for each mixture, assuming the assay value of the control mixture is 100% (Residual FVIII% = FVIII level of patient dilution/ FVIII of the control tube x100)
- If the residual FVIII is between 80-100%, the sample does not contain an inhibitor
- <60%= consistent with an inhibitor

7) The lowest dilution of test plasma that gives a residual FVIII level close to 50% (30- 60%) is used to calculate the strength of the inhibitor
8) The BU obtained at a residual FVIII level of ~50% (as read off a standard graph: residual FVIII vs IU) then needs to be multiplied by the dilution factor to determine to actual BU

19
Q

What is the definition on 1 Bethesda unit?

A

1 Bu is defined as the amount of inhibitor in a plasma sample which will neutralise 50% of 1 unit of factor VIII in normal plasma after a 2hr incubation at 37°C.

20
Q

What is a ‘D-dimer’ and what are the 3 most common methods of measuring D-dimers?

A
  • Specific fibrin degradation product
  • Generated when cross-linked fibrin is degraded by plasmin
  • Greater specificity for fibrin (as opposed to fibrinogen)

Methods:

1) Latex agglutination using a bispecific antibody conjugate with binding sites for the latex particle and D-dimer
2) ELISA assays using an antibody specific for D-dimer
3) Whole blood agglutination assay that uses a bi-specific antibody conjugate with binding sites for both D-dimer and an antigen on the red cell.

21
Q

What are some of the limitations of using a D-dimer assay?

A
  • Not specific for DIC and large vessel thrombosis: increased in inflammatory and malignant states
  • False positives reported with rheumatoid factor, red cell haemolysis, lipaemia and an elevated bilirubin
  • Spurious D-dimer concentrations due to heterophile antibodies have also been reported in the literature. Heterophile blocking agents or use of an alternative method can overcome this problem.
  • Levels increase in pregnancy and are linked to gestational age. This reduces the tests sensitivity and specificity for VTE in pregnancy.
  • D-dimer levels increase with age which reduces the sensitivity and specificity for VTE in the elderly
22
Q

Which tests are affected and which are generally not affected by the new anticoagulant drugs?

A
  • Tend to affect all tests that measure clotting time

- Latex agglutination, immunoassays, chromogenic assays (i.e. Protein C) generally are not affected.