Version 1
: Received: 7 March 2023 / Approved: 8 March 2023 / Online: 8 March 2023 (03:04:37 CET)
How to cite:
Kawase, T.; Suzuki, K.; Kamimura, M.; Mochizuki, T.; Ushiki, T. Optimized Protocol for Preservation of Human Platelet Samples for Fluorometric Polyphosphate Quantification. Preprints2023, 2023030143. https://doi.org/10.20944/preprints202303.0143.v1.
Kawase, T.; Suzuki, K.; Kamimura, M.; Mochizuki, T.; Ushiki, T. Optimized Protocol for Preservation of Human Platelet Samples for Fluorometric Polyphosphate Quantification. Preprints 2023, 2023030143. https://doi.org/10.20944/preprints202303.0143.v1.
Cite as:
Kawase, T.; Suzuki, K.; Kamimura, M.; Mochizuki, T.; Ushiki, T. Optimized Protocol for Preservation of Human Platelet Samples for Fluorometric Polyphosphate Quantification. Preprints2023, 2023030143. https://doi.org/10.20944/preprints202303.0143.v1.
Kawase, T.; Suzuki, K.; Kamimura, M.; Mochizuki, T.; Ushiki, T. Optimized Protocol for Preservation of Human Platelet Samples for Fluorometric Polyphosphate Quantification. Preprints 2023, 2023030143. https://doi.org/10.20944/preprints202303.0143.v1.
Abstract
Platelet polyphosphate (polyP) can be conveniently quantified by 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, possible protocols were screened, and passed 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 fixed with ThromboFix for 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. In platelet suspension maintained in fixed conditions at 4 °C (control), platelet polyP levels significantly decreased by 20% at and after 7 days. In contrast, platelet polyP levels in both P-PRP and washed platelet suspensions were maintained without significant reduction for up to 6 weeks by removing ThromboFix after fixation and subsequent freezing in pure water at −80 °C. Owing to the low specificity of DAPI binding and the wavelengths used, fluorometric polyP quantification is often interfered with. Our validated protocols will enable long-term preservation and high-throughput polyP quantification and could be applied to relatively large cohort studies.
Keywords
platelets; polyphosphate; preservation
Subject
LIFE SCIENCES, Biochemistry
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
