Whole blood was collected from healthy donors according to the German guidelines for hemotherapy with written informed consent. The study was approved by the ethical board of the Universitätsmedizin Greifswald, Germany. Pooled platelet concentrates from whole blood buffy coats were produced according to the standard method for producing therapeutic platelet concentrates. In brief, after centrifugation (4000 g, 10 min), whole blood in citrate phosphate dextrose solution (CPD, Macopharma, France) was separated into red cell concentrate, plasma, and buffy coat. Buffy coats of 4 blood group identical donations were pooled, and 250 mL additive solution SSP+ (Macopharma) was added. SSP+ itself contains 2 mM magnesium. By centrifugation (720 g, 15 min), platelets were separated from residual red blood cells and leukocyte depleted (LEUCOFLEX® LXT Filter, Macopharma). PC bags were split into six bags (gas permeable bags, Macopharma) and stored under agitation at RT or 4 °C for 7 days.
Platelet concentrate storage
Magnesium sulfate solution (Inresa Arzneimittel GmbH, Germany) was added to four of the six bags in increasing concentrations (Table 2). PC was stored under agitation on an orbital shaker (LPR1, Melco Engineering Corp., USA). Sampling took place on day 0 (production day), day 1, day 4 and day 7.
In vitro platelet function testing
The ability of platelets to respond to a hypotonic environment was determined as hypotonic shock response (HSR) by light transmission aggregometry (LTA).37 Platelets were exposed to distilled water or 0.9% w/v sodium chloride solution as a control. Due to the osmotic gradient, the water diffuses into the platelets, which leads to their swelling. As a result of swelling, the refractive index of platelets increases, resulting in a decrease in light transmission. The percentage of HSR was calculated as (T2-T3)/(T2-T1)×100, with T1= transmission of platelet suspension in sodium chloride; T2= transmission of platelets in distilled water; T3= “plateau” transmission value after 4 min of platelets incubated in distilled water.
Platelet activation was determined by CD62P expression before and after the addition of thrombin receptor activating peptide 6 (TRAP-6). 3x108/mL platelets were incubated for 15 min at 37 °C with 20 µM TRAP-6 (Hart Biologicals, UK) or PBS buffer (w/o Ca2+, Mg2+; pH 7.2) as the negative control, fixed 20 min with 0.5% paraformaldehyde (Morphisto Laborchemikalien, Germany) and washed twice (2 mL PBS; 650 g, 7 min, RT). The pellet was resuspended in 500 µL PBS and analyzed by flow cytometry (FC500, Beckman Coulter, USA). Platelets were gated using a mouse-anti-human CD41-PeCy5 (clone P2, Beckman Coulter) labeled monoclonal antibody. The increase of CD62P exposure on CD41-positive events was determined using CD62P-FITC (clone CLBThromb/6, Beckman Coulter, USA). The mean fluorescence intensity of TRAP-6 activated platelets is given as fold increase in comparison to the respective buffer controls.
Platelet aggregation was analyzed over 7 min at 37 °C in a 4-channel-aggregometer (DiaSys, Germany) after the addition of 8 μg/mL collagen (Mölab, Germany).
Whole blood as a matrix for ex vivo quality control of platelet concentrates
To simulate in vivo conditions, we prepared platelet-depleted whole blood (dWB). Whole blood was collected from a healthy donor according to the German guidelines for hemotherapy, using a bag system containing citrate-phosphate-derivative with adenine (CPDA) solution for anticoagulation. Directly after donation, whole blood was leukocyte and platelet depleted by a LEUCOFLEX® LXT leukocyte depletion filter, which removes 99.999% of leukocytes and platelets (Macopharma, France). Cell counts were determined before and after filtration by an automated blood cell analyzer (Sysmex XP300, Sysmex Deutschland GmbH, Germany). Depleted whole blood was stored at 4 °C until use. Platelets stored at 4 °C at different Mg2+ concentrations were either analyzed directly in platelet storage buffer SSP+ or spiked into depleted whole blood to achieve a final platelet concentration of 300,000/µL and rewarmed at 37 °C for one hour before analyses. For aggregometry studies, whole blood was centrifuged at 120 g for 20 min to obtain PRP.
Biomechanical platelet characterization
RT-DC is a method for the biomechanical characterization of cells with throughput rates of up to 1,000 per second (Supplementary figure S1).23 In brief, platelets are pumped at flow rates of 0.006 mL/s from a reservoir through a narrow channel on a chip assembled on the AcCellerator system (Zellmechanik Dresden). The microfluidic chip consists of a constriction of 15 mm x 15 mm cross-section with a length of 300 mm and is connected to a syringe pump (NemeSys, Cetoni, Germany). Upon entering the channel and during the passage, the cells are subjected to both hydrodynamic shear forces and different pressure gradients. The deformation of platelets caused by these external forces are recorded by a high-speed camera. The individual images are then analyzed with an evaluation algorithm (ShapeOut, version 0.8.6, Zellmechanik Dresden) for the deformation of the individual platelet (Supplementary Figure S1).17 Samples were taken on days 1, 4, and 7 after PC production. For RT-DC 50 mL of platelets in SSP+/plasma were diluted in Carrier B (Zellmechanik Dresden; 0.6% (w/v) methylcellulose in PBS, without Ca2+ and Mg2+) to a final concentration of approximately 1x107/mL. Calculations are performed at different flow rates representing different experimental conditions.
Analysis of cytoskeleton proteins by immunofluorescence microscopy
Platelets from PC of all 6 storage conditions were adjusted to a platelet count of 50,000 platelets/µL each with 2 % paraformaldehyde in PBS (w/o Ca2+, Mg2+, pH 7.2) and incubated for 15 minutes at RT. At the end of the incubation period, 100 µL of each sample was cytospin centrifuged by for 5 minutes at 700 rpm on microscopic slides. Following centrifugation, the slides were washed three times for 5 minutes at RT with PBS (w/o Ca2+, Mg2+, pH 7.2) and stored at -20 °C. The cells were treated with permeability buffer (0.1 % Triton-X in PBS and BSA 2 %) for 10 minutes at RT in a humid chamber, and cells were then washed twice with 50 µL PBS for 10 minutes at RT. Platelets were stained for α-tubulin (mouse anti-human α-tubulin IgG, Sigma-Aldrich, St. Louis, Missouri, USA) at RT for at least one hour, washed again twice with 50 µL PBS and then incubated with 50 µL secondary donkey anti-mouse IgG Alexa Flour 568 (Abcam, Cambridge, UK) for one hour at RT, incubated with 50 µL of phalloidin ATTO 488 solution (1:200; ATTO-TEC GmbH, Siegen, Germany) for further 30 minutes at RT in the dark and then washed twice with PBS. Fluorescence microscopy was performed on a Leica SP5 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany) equipped with HCX PL APO lambda blue 40.0x/1.25 OIL UV objective. For image acquisition, fluorescent tags Atto-488 and AlexaFluor-568 were excited by argon (488nm) and helium-neon (HeNe) 563nm laser lines, respectively, selected with an acousto-optic tunable filter (AOTF). Fluorescence emission for Atto-488 and AlexaFluor-568 was collected between 505-515 nm and 550-570 nm on hybrid detectors (HyD) and photomultiplier tubes (PMTs), respectively. Assessment of distribution and organization of marginal band α-tubulin staining was performed by measuring cross-sectional line profile (5 µm length and 1 µm width) of non-saturated grayscale fluorescence intensities (pixel values) of immunofluorescent probes across individual platelets in confocal images using Leica Application Suite X (Version 3.7.1, Leica Microsystems, Wetzlar, Germany). Data were plotted using GraphPad Prism version 8.0.0 for Windows, (GraphPad Software, San Diego, California USA). For each storage condition, the microscopic images of at least 10 single platelets were analyzed. In addition, whole blood spiked with platelets from stored PC was also used to prepare blood smears, which were stained as described above. Here, an Olympus BX 40 microscope with UPlanSApo 60x/1.35 Oil objective was used with software cellSensStandard (Imaging Software cellSens, Olympus Corporation, Tokyo, Japan).
Desialylation of stored platelets
To evaluate the effect of Mg addition on desialylation of platelets during storage at 4 °C we determined the binding of fluorescein isothiocyanate (FITC) labeled Erythrina cristagalli lectin (ECL) and Ricinus communis lectin agglutinin (RCA) to platelet surface glycoproteins of RT and cold stored platelets with or without Mg2+ addition. PC was adjusted to a cell count of 300,000/µL and labeled by CD61-AlexaFluore647 platelet marker (Biolegend, USA). FITC labeled RCA and ECL were added (1 µL 1:500 RCA or 1:250 ECL in SSP+, Biozol, USA) and incubated in the dark for 20 min. After that 200 µL SSP+ was added, and the samples were washed (650xg, 7 min). The binding of ECL and RCA was analyzed using an FC500 flow cytometer (Beckman Coulter, USA). As a positive control, sialidase (neuraminidase, Sigma Aldrich, USA) was used.