PB1091 - A potential suitable alternative to Triton-X-100 for use in sample preparation for platelet nucleotide analysis

Background: The nucleotides ATP and ADP present in two distinct pools within platelets. The metabolic pool accounts for approximately 40% of the nucleotide population maintained in a ratio of 8:1 ATP: ADP. The remaining 60% are found in the platelet dense/delta granules in a ratio of 2:3 ATP:ADP and play a role in activation and propagation of platelet aggregation in response to haemostatic challenge. Reduction, absence and/or altered relative ratio of ATP:ADP within the platelet delta granules are characteristic of platelet storage pool defects and require a robust, sensitive quantitative assay for diagnosis.

Aims: Preparation of platelet lysates for nucleotide analysis has relied on using 20% Triton-X-100 solution for membrane dissolution however this is now commercially unavailable due to it’s ecotoxicity. We have investigated the use of alternative detergents 20% tween 20 and 1% tergitol solutions to assess their potential as a viable replacement.

Methods: Platelet nucleotide analysis relies on direct measurement of ATP in extracted platelet lysates using luciferase enzyme reagent. Platelet ADP content is estimated following it's conversion to ATP, using pyruvate kinase, and subtracting the initial ATP concentration from the total ATP measured.
Paired extracted platelet lysates from normal donor platelet rich plasma using 20% Triton-X-100 and either 20% tween 20 or 1% tergitol were prepared in parallel and assayed for ATP and ADP content.

Results: Figure 1 shows preliminary results from paired samples lysed and extracted using 20% triton-X-100 and 20% tween-20 and show a large discrepancy in measured ATP. Comparison of samples extracted using 20% Triton-X-100 and 1% tergitol showed improved consistency of ATP and ADP results (Figure2).

Conclusion(s): The use of 1% tergitol as an alternative detergent to 20% Triton-X-100 has shown some promise in preliminary studies. It is proposed to expand this data to assess it's suitability for use in the routine diagnosis of platelet storage pool disorders.