56. Jahrestagung der Deutschen Gesellschaft für Transfusionsmedizin und Immunhämatologie

Prof. Dr.

Vorsitz

PD Dr.

Platelet reactivity in platelet concentrates is promoted by short-term refrigeration

Blood Components

Background The use of cold-stored platelets is discussed to be beneficial for the treatment of acute hemorrhage. Recently, we could show that refrigeration for only 1-2 h was sufficient to increase reactivity of fresh platelets, rendering the opportunity to instantly generate platelets with a higher hemostatic capacity. In this study, we investigated the effect of cold storage for 1 h (CT) in stored apheresis-derived platelet concentrates (APC) after foregoing storage at room temperature (RT).Methods APC stored for 1 day or for 2 days at RT, were exposed to cold temperature for 1 h. APC continually stored at RT served as controls. Consecutively, platelet integrity was analyzed by different methods. Inhibitory signaling was explored by vasodilator-stimulated phosphoprotein (VASP) phosphorylation using flow cytometry and by measurement of cyclic nucleotide concentrations. Western Blot analysis was used to investigate activating signaling pathways. Light transmission aggregometry was performed with activators in threshold concentrations.Results After cold exposition, VASP phosphorylation levels were partially decreased in APC at storage day 1 and day 2, compared to continued RT storage. Additionally, induced cGMP and cAMP levels were reduced in CT-stored APC. Basal phosphorylation levels of ERK1/2 kinase or p38 MAP kinase were emphasized after cold storage. Aggregation values induced with threshold concentrations of adenosine diphosphate, collagen or TRAP-6 reached higher values in APC after refrigeration for 1 h. In general, cold-mediated effects were more pronounced in APC, stored for 2 days.Conclusion In APC at storage day 1 and at storage day 2, exposition to cold temperature for 1 h results in increased platelet reactivity, characterized by an attenuation of inhibitory signaling, by supported activating signaling pathways and by facilitated threshold aggregation responses. Compared to permanently RT-stored APC, short-term refrigeration may be an opportunity to manufacture APC with improved hemostatic capacity "on demand", favorable for the treatment of hemorrhage.Offenlegung Interessenkonflikt: The study was supported by the "Stiftung Transfusionsmedizin und Immunhämatologie" of the DGTI. The authors have no other conflicts of interests to declare.

Freier Vortrag

Ex vivo investigations of thrombus formation using platelet concentrates

Background Platelet concentrates (PCs) are used to treat or prevent bleeding in patients with impaired platelet function or after injury. Hemostatic functions of PCs have been investigated intensively under steady state settings but until now no standard methods are available for platelet function tested under shear stress. Our aim is to establish an ex vivo model to evaluate the contribution of PCs in thrombus formation and to investigate the effect of different storage conditions on platelet functions.Methods First, microfluid channels were coated with collagen (0.1 mg/mL) overnight at 4°C and the next day blocked with HSA (human serum albumin, 1%, 1h, room temperature). Next, PCs stored for 24h at room temperature (RT) were incubated with Calcein (4 µM, 15 min, RT) and recalcified with CaCl (7.5 mM) and MgCl (3.75 mM). TRAP (thrombin receptor-activating peptide, 2.5 µM) was added to initiate thrombus formation. Finally, platelet-depleted whole blood samples from healthy donors were spiked-in with PCs and immunofluorescence pictures were taken randomly. Additionally, PCs were stored for 1, 4, 7 and 10 days either at RT or at 4°C. Samples were stained with CD41 antibody (0.5µg/ml), recalcified and applied to the ex vivo system.Results To establish the ex vivo model, platelets isolated from PCs were tested after 24h of storage at RT and cells from a healthy donor were used as control. Platelets from PCs, after reconstitution, showed stable thrombus formed upon incubation with TRAP. On the contrary, cells incubated with buffer did not form any thrombus (% Surface area covered, Buffer vs. TRAP 1.860±8.844 vs. 10.70±2.255, p=0.0172). To evaluate the effect of cold storage on platelet functions we analyzed the extent of thrombus formation of PCs stored at RT and 4°C upon CD41 staining. Our preliminary results indicate that cold-stored platelets tend to form larger clots under flow conditions after TRAP activation compared to PCs stored at RT (Figure 1).Conclusion The results indicate that our ex vivo assay, which simulates PC transfusion in thrombocytopenic patients, is suitable to test the hemostatic functions of PCs under physiological flow conditions. Moreover, it allows to investigate cold-induced effects on platelet functions during storage time.Offenlegung Interessenkonflikt: No conflict of interest.

Towards marketing authorization of isoagglutinin depleted human plasma for blood group independent transfusion

Background Isoagglutinins in plasma require for ABO blood group compatibility. We developed a GMP conform procedure to deplete the isoagglutinin titer of fresh frozen plasma (FFP) units group A, B, and O. The method is based on isoagglutinin adsorption onto added red cells. A requirement for the approval of isoagglutinin depleted plasma from blood group A units was an extensive validation study to be able to assess the impact of the plasma treatment on coagulation and immune parameters.Methods We developed a bag system, which enables pooling of three FFP units, addition of the adequate RCC volume and subsequent removal of isoagglutinin red cell sediment in a closed system. After incubation at 20-24°C for two hours the isoagglutinin red cell sediment was removed from the plasma pool by centrifugation (4,000g, 10min) and separation. Within the validation study we compared plasma units of 12 production processes (=36 units) with or without the addition of RCC for isoagglutinin adsorption with regard to the following parameters: anti-B titer, free hemoglobin, residual red cells, factors II–XIII, fibrinogen, vWF antigen+activity, protein C + S, antithrombin, ADAMTS13, aPTT, Quick value, lactate dehydrogenase and complement activation.Results Starting from maximum values of 1:16 in the plasma pools, total anti-B titers were reduced to &lt; 1:1 in all units of the treatment arm. Free hemoglobin was similar between the control and treatment group (24.8±19.7 µM vs 22.9±15.4 µM; p&gt;0.5). No residual red cells were found by microscope counting. All other coagulation and immune parameters showed no significant differences between the control and treatment arms (Table 1).Conclusion We present a fully automated GMP conform procedure for the production of isoagglutinin depleted plasma without any impact on a large set of coagulation and immune parameters.Offenlegung Interessenkonflikt: Macopharma kindly provided parts of the bag system used.

Indocyanine green labeled platelets for survival and recovery studies

Background Before being implemented in daily clinical routine, new production strategies for platelet concentrates (PC) must be validated for their safety. Besides in vitro testing, the establishment of new methods requires the labeling of platelets for in vivo studies of platelets&quot; survival and recovery. Indocyanine green (ICG) is a FDA and BfArM approved near-infrared (NIR) fluorescent dye suitable for non-radioactive direct labeling of platelets for diagnostics.Methods Platelets from PC in storage solutions with different plasma concentrations were labeled with ICG up to concentrations of 200 µM. Whole blood was used as an ex vivo matrix to monitor the labeling stability of ICG-labeled platelets. The impact of labeling processes was assessed by the quantification of CD62P expression and PAC-1 binding as platelet function markers. Platelet aggregation was analyzed by light transmission aggregometry. ICG labeling efficiency and stability of platelets was determined by flow cytometry.Results Platelets from PC were successfully labeled with 10 µM ICG after 1 and 4 days of storage. The best labeling efficiency of 99.8% ± 0.1% (immediately after labeling) and 81% ± 6.2% (after 24 hours incubation with whole blood) was achieved by plasma replacement with 100% platelet additive solution for the labeling process. The washing process slightly impaired platelet function, but ICG labeling itself did not affect platelets. Immediately after the ICG labeling process, plasma was re-added resulting in a recovered platelet function. Conclusion We developed a Good Manufacturing Practice compatible protocol for indocyanine fluorescent platelet labeling suitable for non-radioactive tracing in survival and recovery studies in vivo.Offenlegung Interessenkonflikt: none

Freier Vortrag (Reinhold-Eckstein-Investigator-Award)

Univ.-Prof. Dr.

Transcription factors STAT3 and MYC mediate human platelet lysate-induced cell proliferation

Background Human platelet lysate (HPL) has emerged as an efficient cytokine and growth factor providing medium supplement, replacing fetal bovine serum (FBS) for ex vivo clinical cell expansion. As growth promoting effects of HPL may be based on intracellular mechanisms, we hypothesized a specific influence on cell cycle target gene expression and aimed to identify the main molecular key players.Methods Cell cycle target gene expression of bone marrow-, umbilical cord- and white adipose tissue-derived stromal cells cultured in HPL- or FBS-supplemented media was compared using RT-qPCR and a cell cycle specific antibody array. As HPL is rich in growth factors that are ligands of tyrosine kinase receptor (TKR) pathways, we applied TKR inhibitors and analyzed cell proliferation. Additionally, genome profiling, RT-qPCR and Western blotting were used to identify downstream mediators of HPL-derived growth signals.Results RT-qPCR as well as cell cycle specific antibody array revealed significant upregulation of cell cycle genes in stromal cells cultured in HPL. Blocking specific TKR pathways lead to significantly reduced cell proliferation. In response to HPL we could detect an enhanced expression of the transcription factors signal transducer and activator of transcription 3 (STAT3) and MYC, both known as TKR downstream effectors and stimulators of cell proliferation. Furthermore, specific inhibition of STAT3 resulted in a significant reduction of HPL-induced cell proliferation and cell cycle gene expression.Conclusion Based on our results we suppose that HPL-promoted cell proliferation is induced by enhanced TKR signaling and thus elevated expression of the TKR downstream effectors STAT3 and MYC. The enhanced expression of STAT3 and MYC might in turn stimulate the expression of cell cycle promoting target genes.Offenlegung Interessenkonflikt: The authors declare no conflict of interest.

Artificial oxygen carriers optimized for ex situ organ perfusion

Background Normothermic machine perfusion relies on sufficient O2 supply, while availability of blood by the donor is limited. Artificial oxygen carriers (AOC) based on perfluorocarbons (PFC) can replace blood, as they are synthetic, fully halogenated alkanes, physically dissolving respiratory gases &amp; are of unlimited availability. PFCs must be emulsified for use in aqueous media, which rises stability issues.[1]Methods A new type of PFC-based AOC was developed by using an emulsifier combination of albumin &amp; lecithin. A special high pressure homogenizer (Microfluidizer™) with ice cooling was used to synthesize the novel lecithin-modified nanoscale oxygen carriers with a perfluorodecalin core (LENOX).[2] LENOX were synthesized in water, Custodiol™ and STEEN Solution™. The applicability of LENOX for machine perfusion was tested by circulating LENOX in an ex situ perfusion circuit without organ at 37 °C for 4 h. Samples were taken at regular intervals and analyzed by e.g. dynamic light scattering (size), rheometry (viscosity) &amp; a blood gas analyzer (pH, O2 release, free ions). Error bars shown in the figures represent standard deviation of n=3 experiments.Results LENOX could be synthesized in all three media and formed a sufficiently stable emulsion. The particle size analysis showed a similar behavior for LENOX in water &amp; Custodiol™. LENOX in STEEN Solution™ revealed significantly larger mean particle diameter than the other media. However, LENOX in all 3 media were stable over 240 min &amp; small enough for use in machine perfusion settings (Fig. 1). All three variants of LENOX showed the same amount of O2 release, which, due to non-existent consumption, reached a maximum of about 320 hPa (240 mmHg) after 60 min. The different perfusion media depicted no influence on the functionality of LENOX (Fig. 2).Conclusion The novel AOC LENOX revealed high stability despite the difficult requirements of organ perfusion. Compatibility with clinically relevant perfusion media such as Custodiol™ or STEEN Solution™ makes LENOX an interesting alternative to blood for organ perfusion.Offenlegung Interessenkonflikt: Three of the named authors are listed as inventors in the given patent

Blood Components

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