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

AI Article Synopsis

  • Researchers developed methods to quantify platelet polyphosphate (polyP) using a fluorescent dye called DAPI, but standard preservation techniques for biological samples are still lacking.* -
  • In their study, they tested different protocols for preparing samples from healthy male donors, finding that platelet polyP levels dropped significantly in fixed samples after a week, while other methods preserved levels for up to six weeks.* -
  • The study highlights the challenges of accurate quantification due to DAPI's low specificity and presents validated protocols that can enhance the preservation and analysis of polyP in larger studies.*

Article Abstract

Platelet polyphosphate (polyP) can be conveniently quantified by exploiting a recent methodological breakthrough using 4',6-diamidino-2-phenylindole (DAPI). However, the preservation of these biological samples has not yet been standardized. In a preliminary study, potential protocols were screened, while accepted protocols were further tested in this study. Pure-platelet-rich plasma (P-PRP) samples and washed platelet suspensions were prepared using blood obtained from non-smoking healthy male donors and were fixed with ThromboFix for 20-24 h at 4 °C. Mass polyP levels were determined using a fluorometer at wavelengths of 425 and 525 nm. Platelet polyP levels were normalized to platelet counts. Statistical analyses were performed using non-parametric tests. Platelet polyP levels significantly decreased by 20% after 7 days in the platelet suspension maintained under fixed conditions at 4 °C (control). In contrast, the platelet polyP levels in both the P-PRP and washed platelet suspensions were maintained without a significant reduction for up to 6 weeks by removing ThromboFix after fixation and subsequent freezing in pure water at -80 °C. Fluorometric polyP quantification often interferes with the low specificity of DAPI binding and the wavelength used. Our validated protocols will enable long-term preservation and high-throughput polyP quantification and can be applied to relatively large cohort studies.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10366864PMC
http://dx.doi.org/10.3390/mps6040059DOI Listing

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