Irreversible oxidations of platelet proteins after riboflavin-UVB pathogen inactivation.

Pathogen inactivation technologies are known to alter in vitro phenotype and functional properties of platelets. Because pathogen inactivation generates reactive oxygen species, oxidative stress is considered as one of the plausible cause at the origin of the platelet storage lesion acceleration after treatment. To date proteomics has been used to document the protein variations to picture out the impact. Here, platelet concentrates were prepared from buffy-coats in Intersol additive solution, leukoreduced and pathogen inactivated using a riboflavin/UVB treatment. At day 2 of storage the platelet proteomes of control (untreated) and treated platelet concentrates were investigated against the site specific oxidation by liquid chromatography coupled to tandem mass spectrometry in a shotgun experiment. The shotgun approach detected 9350 peptides (and 2534 proteins) of which 1714 were oxidized. Eighteen peptides were found exclusively oxidized in treated platelets whereas 3 peptides were only found oxidized in control. The present data evidenced an interference with several proteins involved in platelet aggregation and platelet shape change (such as talin and vinculin).

Towards the understanding of the UV light, riboflavin and additive solution contributions to the in vitro lesions observed in Mirasol®-treated platelets.

OBJECTIVES: Pathogen reduction technologies are implemented to increase the safety of blood products. We previously showed that the UVB alone significantly contributes to the storage lesions observed in platelets treated with riboflavin/UVB using a home-made illuminator. The present study aims at confirming these observations using the commercial Mirasol® technology. METHODS: A three-arm study (untreated, UV-, Mirasol®-treated platelets) was conducted to investigate the platelet storage lesions throughout storage (n=4). A two-arm study was then designed to compare Intersol and T-PAS+ additive solutions (n=3). Phenotype and functional platelet characteristics were assessed using flow cytometry, aggregometry, antioxidant assays and metabolic parameters. RESULTS: Mirasol®-treated platelets exhibit enhanced storage lesions compared to controls (increase of activation markers and glycolysis rate, lower hypotonic shock and double-agonist activation responses, and decrease of total antioxidant capacity). Here, we also confirmed that the UV radiation alone is causing platelet lesions. Riboflavin tends to have an intracellular protective role while it decreases the extracellular antioxidant defenses. Furthermore, benefits of platelet additive solutions containing potassium and magnesium were confirmed as it reduces the extent of storage lesions. CONCLUSIONS: The photosensitizer, UV illumination and composition of the platelet additive solutions are key parameters influencing the platelet storage lesion. The clinical relevance of these findings is not fully understood and recent published clinical studies could not show increase in bleeding in patients receiving Mirasol-treated platelets. New developments in storage solutions might help to improve storage conditions of PRT-treated platelets and should be prioritised as research subject in the future.

The storage lesions: From past to future.

Red blood cell (RBC) concentrates are stored in additive solutions at 4oC for up to 42 days, whereas platelets concentrates (PCs) are stored at 22oC with continuous agitation for up to 5 to 7 days, according national regulations, and the use or not of pathogen inactivation procedures. Storage induces cellular lesion and alters either RBC or platelet metabolism, and is associated with protein alterations. Some age-related alterations prove reversible, while other changes are irreversible, notably following protein oxidation. It is likely that any irreversible damage affects the blood component quality and thus the transfusion efficiency. Nevertheless, there still exists a debate surrounding the impact of storage lesions, for both RBCs and PCs. Uncertainty is not completely resolved. Several studies show a tendency for poorer outcomes to occur in patients receiving older blood products; however, no clear significant association has yet been demonstrated. The present short review aims to promote a better understanding of the occurrence of storage lesions, with particular emphasis on biochemical modifications opening discussions of the future advancement of blood transfusion processes. The paper is also an advocacy for the implementation of an independent international organization in charge of planning and controlling clinical studies in transfusion medicine, in order to base transfusion medicine practices both on security principles, but also on clinical evidences.

Automatic washing of thawed haematopoietic progenitor cell grafts: a preclinical evaluation.

BACKGROUND AND OBJECTIVES: Autologous haematopoietic progenitor cell (HPC) is a prerequisite for high-dose chemotherapy in treatment of several haematologic and non-haematologic malignancies. HPCs are collected by apheresis and cryopreserved until infusion. Postinfusion adverse events have been in part related to the dimethyl sulphoxide (DMSO) used as cryoprotectant. The aim was to evaluate (i) an automated sequential washing procedure for DMSO removal in thawed HPC grafts and (ii) washing solutions in replacement of hydroxyethyl starch (HES)-based solutions. MATERIALS AND METHODS: A total of 26 HPC bags cryopreserved with 10% DMSO and intended for disposal were used. The Sepax-2 (Biosafe, Eysins, Switzerland) was evaluated using a sequential washing procedure. Outcomes were CD34+ cell recovery and viability after washing. RESULTS: The Ringer lactate supplemented or not with albumin 2·5-5% presented satisfactory results compared with HES solution in terms of CD34+ cell recovery and viability after washing. However, the apparition of aggregates led us to renounce to these alternative solutions. Using HES solution and a sequential washing of three bags, we showed the elimination of 95% of the DMSO, a postwash viable CD34+ cell recovery of 79·9 ± 9·4% and a total nucleated cell viability of 66·5 ± 9·3%. CONCLUSION: The preclinical evaluation of an automated sequential washing procedure for DMSO removal in thawed HPC grafts has proven to be effective and comparable to previously published data. Despite our attempt to find an alternative solution to the HES solution, more efforts should be done on this side to reach a consensus on cryopreservation protocols.

Receptor maturation and synaptogenesis in the golden hamster cochlea.

Maturation of hamster cochlea was studied using light and electron microscopy. Critical stages of receptor and neural structure development have been determined. At birth the hamster cochlea shows a pronounced immaturity, but innervation can already be found. 2 or 3 days later, characteristic afferent synapses can be recognized beginning at the inner hair cell level. Similarly, efferent endings first appear on the inner side at the end of the first week. The onset of auditory function must be related to structures depicted at around 10 days, and cochlear maturation is achieved at about 25 days. The sequence of synaptic development in the cochlea is discussed regarding the general morphogenesis of synapses within the nervous system. Some determinations of the timing of peripheral myelination are given. This process begins almost a week before the presumable date of the onset of function.

[Mauration of unitary responses to tonal stimulation in the cochlear nerve of the kitten]. / Maturation des réponses unitaires à la stimulation tonale dans le nerf cocléaire du chaton

The maturation of single auditory nerve fiber responses to long-duration (500 ms) tone-bursts was studied in kittens at various stages after birth. Spike discharges were examined as a function of three criteria. 10 Latency. Mean value of the "on" peak latency was about 25 ms at 1 to 3 days after birth. It then regressed, reaching 8 to 12 ms at the end of the first week, and 2 to 4 ms at 20 days. 20 Peristimulus histograms (PSTH). Evolution of PSTH revealed the characteristic sequence of unit reactivity to long duration stimuli (i.e. on, rhythmic, and continuous responses). Rhythmic responses is assumed up to now to be related to the immaturity of synaptic junctions below the hair cells. 30 interval histograms. The interspike interval histograms of the discharges showed a similar evolution. A bimodal distribution corresponding to the rhythmic mode of reactivity, appeared at the end of the first week.