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Phospholipid Assay Procedure

This procedure is adapted from Stevenson, KJ “Studies on the development of thromboplastin reagents. A thesis submitted to the Council for National Academic Awards in support of an application for the degree of Doctor of Philosophy,” July 1990. Leicester Polutechnic in collaboration with the UK Reference Laboratory for Anticoagulant Reagents and Control. Withington Hospital, Manchester, UK.

APTT reagents have very disparate phospholipid levels. Some APTT reagents will have very large amounts of phospholipid and some very little. Our APTT reagents have an optimal amount of phospholipid. For SpeckTin-LA™ Lupus Anticoagulant Titration Test sets our phospholipid is checked by the following procedure.

Principle: Phosphatidyl-R type phospholipids form a chloroform soluble complex with ammonium ferro thriocynate

Reagent: Dissolve 27.03 G. ferric chloride hexahydrate and 30.4 G. ammonium thiocyanate in one liter distilled water and mix. This reagent is stable for six months at room temperature.

Procedure Outline:

1. Mix 2.0 ml chloroform, 0.1 ml sample and 2.0 ml ferrithiocyanate reagent.
2. Vigorously mix on vortex mixer for exactly 60 seconds.
3. Centrifuge to separate layers. Remove lower chloroform layer with a glass transfer pipette.
4. Prepare a series of phosphatidylcholine standards in chloroform in the appropriate ranges.
5. Perform steps 1 through 3 with each standard and chloroform blank.
6. Read absorbance at 488 nm of each standard and test against chloroform blank.
7. Plot absorbance of standard vs. concentrate on linear graph paper.
8. Obtain concentration of test sample from standard curve.

 

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