Optimized Protocol for Preservation of Human Platelet Samples for Fluorometric Polyphosphate Quantification.
Methods Protoc
; 6(4)2023 Jun 22.
Article
em En
| MEDLINE
| ID: mdl-37489426
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Tipo de estudo:
Observational_studies
Idioma:
En
Revista:
Methods Protoc
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
Japão