Platelets retain function and can be stored following disruption of human leucocyte antigens.

BACKGROUND AND OBJECTIVES: Antibodies to human leucocyte antigen (HLA) Class-I antigens can lead to refractoriness to platelet transfusion. Although this can be overcome by transfusion of HLA-compatible platelets, they are not always available. Disruption of HLA antigens on platelets by acid treatment may be a suitable alternative when no other components are available. The aim of this study was to assess the effect of HLA disruption and subsequent storage of platelet components. MATERIALS AND METHODS: Platelet components were treated with 0.9% saline or citric acid solution (pH 3.0), and then stored until expiry (Day 7). HLA and platelet glycoprotein expression, platelet viability, activation and sialylation were measured by flow cytometry. Release of soluble factors was measured by ELISA and metabolism by biochemistry analyser. Reactivity to patient anti-sera containing anti-HLA antibodies was measured using platelet immunofluorescence tests (PIFTs) and monoclonal antibody immobilization of platelet antigen (MAIPA) assays. Platelet function was measured using aggregometry and thromboelastography (TEG). RESULTS: Acid treatment reduced detection of HLA Class-I on platelets by 75%, with significant reductions in reactivity to patient anti-sera. Acid treatment reduced platelet content and viability, increased platelet activation and accelerated metabolism. Glycan cleavage was increased by acid treatment. Treatment reduced platelet activation following agonist stimulation by ADP and TRAP-6, but platelets remained functional, displaying increased aggregation response and reduced time to clot formation by TEG. CONCLUSION: Although HLA disruption had some detrimental effects, acid-treated platelets remained functional, retaining their capacity to respond to agonists and form clots, and with further development could be used to support refractory patients.

Donor citrate reactions influence the phenotype of apheresis platelets following storage.

BACKGROUND: Platelet collection and processing methods, as well as donor attributes, can influence platelet function and quality during ex vivo storage. In this study, activation and procoagulant responses in platelets collected from donors experiencing a citrate reaction (CR) were investigated. STUDY DESIGN AND METHODS: Apheresis platelet components (n = 54) were stored in 100% autologous plasma and tested on days 1 and 5 post-collection. Platelet components were categorized into two groups according to whether the donor had experienced a CR during donation (n = 10; non-CR group, n = 44). Platelet aggregation was initiated with collagen and thrombin. Platelet phenotype was characterized by flow cytometry. Fibrinogen binding was assessed following collagen + thrombin stimulation (COATed platelets), and procoagulant activity was assessed using a procoagulant phospholipid assay (PPL). Platelet microparticle (PMP) subsets were enumerated by flow cytometry. RESULTS: Basal von Willebrand factor (VWF) binding was higher in the CR donations when compared with the non-CR group. Collagen aggregation was significantly higher in platelets from CR donations, in contrast to aggregation induced by thrombin. The proportion of phosphatidylserine (PS) positive PMP and PPL clotting time were higher in the CR group, in contrast to the number of basal PS+ platelets and COATed platelets following stimulation. CONCLUSION: Platelets donated by donors who experienced a CR during donation had higher platelet activation response and possibly a more procoagulant PMP phenotype, suggesting that this donor reaction might lead to increased platelet activation.

X-irradiation and gamma-irradiation inactivate lymphocytes in blood components.

BACKGROUND: Irradiation of selected blood components is standard practice for the prevention of transfusion-associated graft-versus-host disease (TA-GvHD). Currently, gamma-irradiation is the most widely used form of irradiation, but there is an increasing interest in X-irradiation, which is considered to be functionally equivalent and safer. However, there is a paucity of contemporary data regarding the ability of X-irradiation to inactivate lymphocytes in blood components. Therefore, the effect of gamma- and X-irradiation on lymphocyte viability and function in blood components was compared. STUDY DESIGN AND METHODS: Lymphocytes were isolated from venous blood by density gradient centrifugation, spiked into plasma/SSP+ to simulate a blood component, and either gamma- or X-irradiated. The phenotype of the isolated lymphocytes was confirmed. Lymphocyte viability was measured using a LIVE/DEAD assay, and function was assessed using mixed lymphocyte culture and CD69 expression post-phorbol-12 myristate 13-acetate (PMA) stimulation. RESULTS: Lymphocyte viability and CD69 expression following PMA stimulation were significantly reduced by both gamma-irradiation and X-irradiation in simulated blood components. Allorecognition and allostimulation were also significantly reduced by both gamma-irradiation and X-irradiation. CONCLUSION: Lymphocyte viability and function are reduced to a similar extent by gamma- and X-irradiation in simulated blood components. As such, X-irradiation is suitable for the irradiation of blood components and, in terms of lymphocyte inactivation, could be used instead of gamma-irradiation.

The role of neuraminidase 1 and 2 in glycoprotein Ibα-mediated integrin αIIbß3 activation.

Upon vascular injury, platelets adhere to von Willebrand Factor (VWF) via glycoprotein Ibα (GPIbα). GPIbα contains many glycans, capped by sialic acid. Sialic acid cleavage (desialylation) triggers clearance of platelets. Neuraminidases (NEU) are responsible for desialylation and so far, NEU1-4 have been identified. However, the role of NEU in healthy platelets is currently unknown. Aim of the study was to study the role of NEU1 and NEU2 in platelet signalling. Membrane association of platelet attached glycans, NEU1 and NEU2 was measured following activation with agonists using flow cytometry. Adhesion on fibrinogen, aggregation and fibrinogen-binding were assessed with/without the NEU-inhibitor, 2-deoxy-2-3-dide-hydro-N-acetylneuraminic acid. Cellular localisation of NEU1 and NEU2 was examined by fluorescence microscopy. Desialylation occurred following GPIbα-clustering by VWF. Basal levels of membrane NEU1 were low; glycoprotein Ibα-clustering induced a four-fold increase (n=3, P<0.05). Inhibition of αIIbß3-integrin prevented the increase in NEU1 membrane-association by ~60%. Membrane associated NEU2 increased two-fold (n=3, P<0.05) upon VWF-binding, while inhibition/removal of GPIbα reduced the majority of membrane associated NEU1 and NEU2 (n=3, P<0.05). High shear and addition of fibrinogen increased membrane NEU1 and NEU2. NEU-inhibitior prevented VWF-induced αIIbß3-integrin activation by 50% (n=3, P<0.05), however, promoted VWF-mediated agglutination, indicating a negative feedback mechanism for NEU activity. NEU1 or NEU2 were partially co-localised with mitochondria and α-granules respectively. Neither NEU1 nor NEU2 co-localised with lysosomal-associated membrane protein 1. These findings demonstrate a previously unrecognised role for NEU1 and NEU2 in GPIbα-mediated and αIIbß3-integrin signalling.