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I. Marini et al.
in comparison to RT-stored PLTs,12-14 while other investi- gators reported that PLTs stored at 4°C can survive in the circulation for several days.15,16
In this context, the residual plasma content of PLT stor- age media may be relevant. Early studies have investigat- ed cold storage of PLTs in plasma and have reported poor recovery and survival.17 Based on these studies, the con- cept of cold storage had been abandoned in routine clini- cal practice. Recently, with the availability of PLT storage in additive solutions (PAS), the cold storage of PCs has seen something of a renaissance. Storage in PAS was sug- gested to maintain better PLT quality and provide protec- tion from storage lesions with the possibility of prolong- ing PC shelf life.18-20 However, it is still unclear whether reduced plasma content improves cold storage of PCs. Furthermore, it is not known whether recovery and sur- vival of PLTs are better after cold storage in PAS compared to storage at RT.
In this study, we investigated the impact of different residual plasma concentrations in apheresis-derived platelet concentrates (APCs) stored at 4°C or at RT. We aimed to clarify whether plasma has protective or detri- mental effects on cold-stored PLTs. Moreover, we assessed in vitro PLT quality and function in APCs to define the opti- mal balance between cold storage in plasma and additive solution. We then initiated a validation study of cold- stored APCs produced under Good Manufacturing Practice (GMP) conditions with 35% residual plasma to verify the feasibility of PLT cold storage for clinical use.
Methods
Preparation of apheresis platelet concentrates
Apheresis-derived platelet concentrates were collected from healthy volunteers according to the German guidelines for hemo- therapy. Ten individuals donated two units of APCs collected with FENWAL AMICUS (Amicus, Fresenius Kabi, Bad Homburg, Germany) and stored in plasma or in PAS (SSP+, Macopharma, Langen, Germany) at different final plasma concentrations [100% (Plasma-APC), 35% (PAS-35-APC) or 20% (PAS-20-APC)] at 4°C and RT. See the Online Supplementary Methods for further details. Finally, PAS-35-APCs were produced under GMP-conditions (12 healthy male donors) as described,21 and stored at RT and 4°C.
In vivo studies
To assess the survival of PLTs derived from APCs, we used the
NOD/SCID mouse model as described previously.22,23 See the Online Supplementary Methods for further details.
Measurement of glycan changes
Glycan pattern was analyzed by flow cytometer (FC) (Navious, Beckman Coulter) using ricinus communis agglutinin (RCA, 0.5 mg/mL, Vector, Burlingame, CA, USA) which binds beta (β)-galac- tose, as described in the Online Supplementary Methods.
Apoptosis
Platelets from APCs were washed, resuspended with 1 mM CaCl2, and stained with Annexin V-FITC (Beckman Coulter) for 60 minutes (min) at RT. Freshly isolated PLTs were incubated with 10 mM ionomycin (Abcam, Cambridge, UK) and used as positive control. See the Online Supplementary Methods for further details.
Platelet adhesion
Coverslips (Corning, New York, USA) were coated with 100 mg/mL of fibrinogen (Sigma Aldrich, Munich, Germany) or colla-
gen (Horm Collagen-Takeda, Linz, Austria). PLTs from APCs (1x108 PLTs/mL) were seeded on coverslips and incubated for 1 hour (h) at RT with TRAP (0.1 mM, Hart Biologicals, Hartlepool, UK). The adherent cells were fixed with 4% paraformaldehyde (PFA) for 20 min at RT. Images were captured from 5 different microscopic fields/coverslips (x100, Olympus IX73, Tokyo, Japan).
Platelet aggregation
Platelet function was analyzed by FC and light transmission PLT aggregation assay (LTA) using a 4-channel-aggregometer (Labitec, Ahrensburg, Germany). See the Online Supplementary Methods for further details.
Hypotonic shock reaction
Hypotonic shock reaction (HSR) was determined by LTA. Percentage of HSR was calculated as described in the Online Supplementary Appendix.
Statistical analysis
Statistical analyses were performed using GraphPad Prism 7 (La Jolla, USA). A t-test was used to analyze normally distributed results. Non-parametric tests were used when data failed to follow a normal distribution as assessed by the D’Agostino and Pearson omnibus normality test. Group comparison was performed using the Wilcoxon rank-sum test and the Fisher exact test with categor- ical variables. In the case of a small number of experiments (<10), the group comparison was performed using the t-test. P<0.05 was considered statistically significant.
Ethics
All studies involving human subjects were approved by the ethics committees of the University Hospital of Tübingen and the Universitätsmedizin Greifswald. Animal studies were approved by the state animal ethics committees of Baden-Württemberg and Mecklenburg-Vorpommern.
Results
Platelet survival after cold storage at different plasma concentrations
At storage day 7, PLTs were injected into the NOD/SCID mice and the survival of PLTs stored at differ- ent conditions was compared in pairs. To enable statistical analysis between PLTs from different donors, the relative survival of Plasma-APC stored at RT was considered as 1.0. Fewer PLTs were found in the circulation when APCs were stored at 4°C compared to RT, regardless of whether PLTs where stored in 35% residual plasma [PAS-35-APC stored at 4°C vs. RT mean±Standard Error of mean (SEM): 1.05±0.02 vs. 0.63±0.16, respectively, P=0.04; Figure 1A] or in 20% residual plasma (PAS-20-APC 0.58±0.05 vs. 0.34±0.05, respectively, P=0.01; Figure 1B).
Regarding the plasma content, similar survival curves were observed at each temperature when PLTs were stored in 35% plasma compared to 100% plasma (Online
Platelet activation and granule release
Platelets (10x106/mL) were incubated with TRAP (40 mM) for 30 min at 37°C, and fixed with 4% PFA for 20 min at RT. The expres- sion of CD62P (CLB-Thromb/6, Beckman Coulter) and CD63 (CLB-Gran/12, Beckman Coulter) was determined by flow cytom- etry (FC) as well as the conformational changes of glycoproteins (GPs) IIb/IIIa complex by PAC-1-antibody (BD Bioscience).
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