Optimized Protocol for Preservation of Human Platelet Samples for Fluorometric Polyphosphate Quantification.

Area of Science:

• Biochemistry
• Hematology
• Analytical Chemistry

Background:

• Platelet polyphosphate (polyP) quantification is vital in hematological research.
• Existing methods for polyP quantification, such as using 4',6-diamidino-2-phenylindole (DAPI), require standardized sample preservation.
• Current preservation techniques lack standardization, potentially affecting experimental outcomes.

Purpose of the Study:

• To evaluate and validate protocols for the long-term preservation of platelet polyphosphate (polyP).
• To establish reliable methods for maintaining platelet polyP levels for accurate quantification.
• To support high-throughput analysis and large cohort studies in platelet research.

Main Methods:

• Screening of potential preservation protocols for platelet samples.
• Testing of selected protocols using pure-platelet-rich plasma (P-PRP) and washed platelet suspensions.
• Quantification of mass polyP levels using fluorometry and normalization to platelet counts.

Main Results:

• Platelet polyP levels decreased by 20% after 7 days at 4 °C in standard conditions (control).
• PolyP levels remained stable for up to 6 weeks in samples fixed with ThromboFix, then frozen at -80 °C after removing the fixative.
• Validated protocols demonstrated effective long-term preservation of platelet polyP.

Conclusions:

• A novel preservation protocol involving fixation and subsequent freezing effectively maintains platelet polyP levels.
• These validated methods overcome limitations of DAPI-based quantification and enable reliable, long-term storage of platelet samples.
• The established protocols facilitate large-scale, high-throughput studies on platelet polyphosphate.