Evaluation of different processing methods to label platelets for in vivo studies

Objective The goal of this study is to evaluate, based on in vitro parameters, different methods of platelet (PLT) labeling (using biotin) in platelet concentrates (PCs) and their impact on the cell phenotype and functions. This is the first step to determine the appropriate processing to label PLTs for in vitro and in vivo studies.

Material and Methods In order to screen three different methods, 45mL from pooled buffy-coat PCs (39% plasma + additive solution) were transferred into 150-mL bags as preparation for the biotinylation process. Methods (1) and (2) were prepared as follows: PCs were washed (by centrifugation) once for condition (1) or twice for condition (2) to remove plasma before the biotinylation and to remove biotin reaction products afterwards. No washing step was applied for method (3): 10mL of the 45mL of PCs were biotinylated and added to the remaining 35mL of non-biotinylated PLTs. All previous conditions were compared with control PLTs (C; no processing and storage in small bags), analyzing the relative expressions of P-selectin, CD42b, and phosphatidylserine expression by FACS during 8 days of storage. All conditions shared the same biotinylation reactant: 1mg [for (1) and (2)] or 1.5 mg [for (3)] of Sulfo-NHS-SS-Biotin (A39258) were diluted in DMSO and SSP+ at 1mg/mL, prior to incubation for 30min at 22°C under agitation. Experiments were done in duplicate.

Results All different conditions tested showed a stable surface expression of biotin with 93% positive PLTs with 1898 median fluorescence intensity (MFI) for (1), 95%/2018 MFI for (2), and 26%/2171 MFI for (3) after 8 days. As expected, processed PLTs (1) and (2) showed an increase of activation (P-selectin), reaching 88% and 83% of positivity after the process vs (3) and (C) with 27% and 21%. This difference was still observed after 8 days with 87%, 62%, 56% and 55% for (1), (2), (3), and (C), respectively.

Conclusion The three conditions of PLTs biotinylation were shown to have different benefits. PLTs in methods (1) and (2) are entirely biotinylated but they are highly activated. In contrast with method (3) only one fraction of PLTs is biotinylated exhibiting the same level of activation as control PLTs (C). This demonstrates that new methods of biotinylation are exploitable. Method 3 will be tested in conventional PC bags where lower levels of lesions are expected due to better storage conditions.

Publication History

Article published online:
18 June 2021