Some adverse reactions to the transfusion of platelet concentrates (PCs) cannot be attributed to antibodies against blood cells or to subclinical microbial agents. It has been suggested that leucocyte-derived inflammatory cytokines such as interleukin (IL)-1, IL-6 and tumour necrosis factor (TNF) may contribute to a larger number of unexplained non-antibody-mediated adverse reactions. Three types of PCs, containing different levels of leucocytes, are currently produced. Filtration is used on demand to further reduce leucocyte contamination of these components. we have monitored the plasma of PCs prepared by the platelet-rich plasma method (PRP), the buffy-coat method or by apheresis for IL-6, IL-1, transforming growth factor-beta (TGF-beta), TNF and interferon gamma (IFN gamma). Biologically active IL-6 increased in stored PRP-PCs from a mean of 140 pg/ml on day 1 to 2395 pg/ml on day 5/6. Elevated levels of IL-8, as detected by immunoassay, were evident in PRP-PCs during routine storage under blood bank conditions. Small amounts of immunoreactive IL-1 with only minimal biological activity were present in some PRP-PCs by day 5/6. No significant increase in the levels of IL-8, IL-6 or IL-1 were seen in buffy-coat PCs during storage for 5/6 d. For apheresis PCs, an increase in IL-8 content, but not in IL-6 over 6 d was observed. In all three types of PCs, elevated amounts of both bioactive and immunoreactive TGF beta were present, but there was no evidence of any biologically active or immunoreactive TNF alpha. Pre-storage filtration of PRP-PCs for depletion of leucocytes prevented the increase in IL-8 and IL-6 levels of these PCs. Our results show that leucocyte reduction by buffy-coat method reduces cytokine levels to a comparable level to filtered or apheresis PCs, containing low levels of leucocytes, but use of these PCs in minimizing the severity and incidence of reactions in recipients will require clinical evaluation. This is the first comprehensive and comparative study which, on the basis of biological activity of cytokines, directly indicates that the mode of platelet production grossly influences the levels of cytokines.

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TRALI is the most common serious adverse effect of blood transfusion in our hospital. Antibodies to HLA class II antigens should be looked for routinely when investigating a possible case of TRALI.

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IntroductionCurrent clinical trials utilize mesenchymal stromal cells (MSCs) expanded in culture, however these interventions carry considerable costs and concerns pertaining to culture-induced losses of potency. This study assessed the feasibility of new clinical-grade technology to obtain uncultured MSC isolates from three human intra-osseous tissue sources based on immunomagnetic selection for CD271-positive cells.Materials and MethodsMSCs were isolated from bone marrow (BM) aspirates or surgical waste materials; enzymatically digested femoral heads (FHs) and reamer irrigator aspirator (RIA) waste fluids. Flow cytometry for the CD45−/lowCD73+CD271+ phenotype was used to evaluate uncultured MSCs before and after selection, and to measure MSC enrichment in parallel to colony forming-unit fibroblast assay. Trilineage differentiation assays and quantitative polymerase chain-reaction for key transcripts involved in bone regeneration was used to assess the functional utility of isolated cells for bone repair.ResultsUncultured CD45−/lowCD271+ MSCs uniformly expressed CD73, CD90 and CD105 but showed variable expression of MSCA-1 and SUSD2 (BM>RIA>FH). MSCs were enriched over 150-fold from BM aspirates and RIA fluids, whereas the highest MSC purities were obtained from FH digests. Enriched fractions expressed increased levels of BMP-2, COL1A2, VEGFC, SPARC and CXCL12 transcripts (BM>RIA>FH), with the highest up-regulation detected for CXCL12 in BM (>1300-fold). Following culture expansion, CD271-selected MSCS were tri-potential and phenotypically identical to plastic adherence-selected MSCs.DiscussionA CD271-based GMP-compliant immunomagnetic selection resulted in a substantial increase in MSC purity and elevated expression of transcripts involved in bone formation, vascularisation and chemo-attraction. Although this technology, particularly from RIA fluids, can be immediately applied by orthopaedic surgeons as autologous therapy, further improvements in MSC purities and pre-clinical testing of product safety would be required to develop this process for allogeneic applications.

These results confirm that Arg35 is important for full expression of S. Pro39 and, surprisingly, Thr3 are also important for full expression of s. Furthermore, Thr3 must be essential for expression of 'N,' as M(v)+ RBCs lack 'N.'

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