ACTIVATED PROTEIN C RESISTANCE UNIVERSITY | Factor V Mutation Screening

Predilution with V-DEF Plasma

The high sensitivity and specificity of the test for the FV:Q⁵⁰⁶ mutation is obtained by prediluting the sample plasma with an excess of V-DEF Plasma. In addition, this allows for analysis of plasma from patients on heparin or oral anticoagulant therapy.

Assay procedure

One volume of citrated and centrifuged sample plasma or Control Plasma is prediluted with four volumes of V-DEF Plasma. The sample is incubated with the APTT reagent for a standard period of time. Coagulation is triggered by the addition of CaCl₂ in the absence and presence of exogenous APC and the time for clot formation is recorded. The two clotting times are expressed as an APC-V ratio calculated as APTT with APC divided by APTT without APC.

Calibration

It is recommended that users establish their own cut-off value for their instrument as described in the kit insert. It should be noted that the APC-V ratio is not equivalent to the APC ratio obtained with the Coatest® APC Resistance kit. Furthermore, the APC-V ratio is generally lower than the APC ratio for the same test sample, independent of instrumentation used.

Results

Factor V-related APC resistance is indicated when the APC-V ratio is below the cut-off value.

Kit Contents

1. V-DEF Plasma, 4 x 4 mL

Stabilized, lyophilized human plasma, with a low level of factor V activity. Contains the heparin antagonist Polybrene®.

2. CaCl₂, 1 x 8 mL

0.025 mol/L in Tris buffer containing 0.5% bovine albumin.

3. APTT reagent, 1 x 16 mL

Purified phospholipid with colloidal silica as contact activator.

4. APC/CaCl₂, 4 x 2 mL

Human APC colyophilized with CaCl₂.

5. Control Plasma Level 1, 1 x 1 mL

6. Control Plasma Level 2, 1 x 0.5 mL

Stability in solution (2–8 °C)

1. V-DEF Plasma, 24 hours

2. CaCl₂, 1 month

3. APTT reagent, 1 month

4. APC/CaCl₂, 5 days

5. Control Plasma Level 1, 6 hours

6. Control Plasma Level 2, 6 hours

Repeatability and precision

APC-V ratios were calculated from 22 single replicate analyses of Control Plasma Level 1 on an ALC instrument using 11 different reagent combinations at 11 different occasions.

APC-V ratio 2.4; CV £ 4% within and between series

HRP = High response plasma

CPL1 = Control Plasma Level 1 (normal response)

CPL2 = Control Plasma Level 2 (low response)

Sensitivity

100% sensitivity for the FV:Q⁵⁰⁶ mutation as determined on Thrombolyzer (n=447), ACL (n=295), ST-4 (n=248) and MLA/Electra (n=50).

Test limitations

Abnormal APTT after predilution with V-DEF Plasma.

Number of determinations per kit

80–160

Catalog #

K823120