Produktinformationen
UNITEST Faktor XII
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This kit is designed for the measurement of factor XII (FXII, Hageman factor) in human plasma. Factor XII is converted to FXIIa with an activator preparation, and the active protease FXIIa cleaves a chromogenic substrate and liberates p-nitroaniline (pNA), which can be measured photometrically¹,².
REAGENTS
The reagents should be stored at 4°C until reconstituted.
1. Factor XII Substrate
10µmol/vial 2AcOH.H-D-CHT-Gly-Arg-pNA, plus mannitol. Dissolve in 10ml sterile distilled water. Stable for at least 6 months at 4°C if kept free from contamination. It may also be stored in aliquots at below -20°C.
2. Factor XII Activator
Dissolve in 5ml distilled water. Stable for 8 hours at 4°C and 6 months at -20°C.
3. Buffer Concentrate
Dilute the vial contents (10ml) with 90ml distilled water. This gives a buffer of 0.05M Tris-HCl, pH 7.9. Store at 4°C.
4. Kallikrein Inhibitor
Dissolve in 10ml distilled water and dilute 1ml with 49ml assay buffer. Stable for 8 hours at 4°C and 6 months at -20°C.
5. Standard Plasma
Add 1.0ml distilled water, leave for 5 minutes at room temperature and then mix gently until completely dissolved. Stable for 8 hours at 4°C.
Reagents required, but not provided: 50% acetic acid; acetone.
BLOOD COLLECTION AND PREPARATION OF PLASMA
Blood (9ml) is mixed with 0.106M Tri-sodium citrate (1ml) and centrifuged at 2000g for 15 minutes at room temperature. The plasma samples should be removed with plastic pipettes within two hours of blood collection and should be assayed immediately or stored frozen at -20°C.
ACETONE TREATMENT OF PLASMA
600µl Standard plasma (or 300µl test plasma) and 200µl acetone (100µl acetone for test plasmas) are pipetted into siliconised glass test tubes (80x10mm), or polypropylene tubes, mixed well and left for 15 minutes at 4°C, then kept on ice until assayed.
PREPARATION OF THE STANDARD CURVE
The acetone treated standard plasma is diluted with buffer as follows:
| Standard % | Plasma (µl) | Buffer (µl) |
|---|---|---|
| 150 | 75 | 325 |
| 100 | 50 | 350 |
| 75 | 37.5 | 363 |
| 50 | 25 | 375 |
| 25 | 12.5 | 388 |
| 0 | Use buffer alone |
Dilute 50µl of each acetone treated test plasma with 350µl buffer.
ASSAY METHOD
Have the substrate and buffer containing kallikrein inhibitor at 37°C. Into siliconised semi-micro cuvettes, siliconised glass or plastic tubes pipette:
| Step | Volume |
|---|---|
| Plasma dilution or buffer | 100µl |
| Add Factor XII Activator | 100µl |
| Mix and incubate at 37°C for 10 minutes, add: | |
| Kallikrein Inhibitor | 300µl |
| Incubate for exactly 1 minute, add: | |
| Factor XII Substrate | 200µl |
| Mix and record the change in optical density per minute at 405nm (rate assay), or incubate for exactly 10 minutes at 37°C, add: | |
| Acetic acid (50%) | 200µl |
| Mix and read optical density at 405nm (end point assay). |
CALCULATION
With the end point assay, if the test plasmas have high bilirubin levels, are lipaemic or have visible haemolysis, blanks must be performed. For the blanks add the reagents in the reverse order and substitute 200µl buffer for the Factor XII substrate and 100µl buffer for Factor XII Activator. The A405 values for the blanks are subtracted from the test values before reading the Factor XII values from the standard curve.
Plot the results as A405 against percentage factor XII for the standard plasma dilutions and read the values for the test plasma from the standard curve. The values can be expressed either as a percentage or in units per ml (U/ml) by applying the formula:
FXII (U/ml) = (% Activity x Potency of Standard*) / 100
* The potency of the standard plasma for FXII (lot UD-0137-0497) is 1.00 U/ml.
PERFORMANCE CHARACTERISTICS
The standard curve should be linear up to 150%. Intra assay cv = 5.5% at 1U/ml.
INTERPRETATION
Normal Range 0.70 - 1.45 U/ml
Factor XII levels may be reduced in congenital deficiency as well as in multi-systems organ failure, sepsis, and DIC³⁻⁵. Factor XII is a component of the contact factor mediated intrinsic pathway of fibrinolysis⁶ and deficiency may be associated with an increased risk of venous thromboembolism⁷⁻⁹. Factor XII levels may be increased in women taking combined oral contraceptives¹⁰⁻¹¹.
HAZARD WARNING
All materials of human origin were tested and found negative for the presence of HBsAG and anti-HIV antibody. However, as with all preparations of human origin, these products cannot be assumed to be free from infectious agents and suitable precautions should be taken in their use and disposal.
NOTE
The recommended standard and test sample dilutions may vary between different batches of this kit owing to differences in the specific activity of some batches of reagents.
REFERENCES
- Gallimore MJ et al. Fibrinolysis 1987; 1: 123-128.
- Walshe K et al. Thromb Res 1987; 47: 365-371.
- Velasco F et al. Thrombos Haemostas 1986; 55: 357-360.
- Martinez-Brotons F et al. Thrombos Haemostas 1987; 58: 709-713.
- Mason JN & Colman RW. Thromb Diath Haemorrh 1971: 26: 325.
- Kluft C et al. Semin Thromb Hemostas 1987; 13: 50-68.
- Goodnough LT et al. Medicine 1983; 62: 248-255.
- Mannhalter C et al. Fibrinolysis 1987; 1: 259-263.
- Lammle B et al. Thrombos Haemostas 1991; 65: 117-121.
- Gorden EM et al. J Lab Clin Med 1980; 96: 781-790.
- Jespersen J & Kluft C. Thrombos Haemostas 1985; 54: 454-459.
CATALOGUE NUMBER: 0068 PRODUCT: Unitest Factor XII Kit
UNICORN DIAGNOSTICS Ltd, London, UK
22.3.94