Blood School.

OBJECTIVES: To describe and evaluate a laboratory-based nursing education activity on transfusion to improve patient safety, an often-neglected opportunity. METHODS: Our transfusion service developed a day-long "Blood School" to provide knowledge, skills, and behaviors to nurses in four aspects of transfusion: blood ordering, sample collection, transfusion procedures, and recognition and reporting of transfusion reactions. We collected survey data on methods and effects of training and hard data on the number of reported patient safety events. RESULTS: Nurses want more hands-on experience to understand transfusion concepts, practice hospital procedures, recognize latent problems, and have behaviors to act effectively. We observed that engagement and understanding are best where participation is highest. Reported patient safety events were lower even as self-reported nursing mistakes increased. CONCLUSIONS: Blood School is a well-received and effective site for nursing education in transfusion. We seek ways to extend and improve laboratory-based nursing training to improve patient safety.

Quality management of a massive transfusion protocol.

BACKGROUND: Massive transfusion is a response to massive uncontrolled hemorrhage. To be effective, it must be timely and address the patient's needs for blood volume, oxygen transport, and hemostasis. STUDY DESIGN AND METHODS: A review was performed on all activations of the massive transfusion protocol (MTP) in a hospital with large emergency medicine, trauma, and vascular surgery programs. Indications, transfused amounts, and outcomes were determined for each MTP event to determine appropriateness of MTP use. Results are presented as descriptive statistics, categorical associations, and simple linear trend relationships. RESULTS: The MTP was activated 309 times in 2016. Of these episodes, 237 were for trauma, 29 for gastrointestinal bleeding, 16 for ruptured abdominal aortic aneurisms, and 25 for a variety of other causes. Trauma-related MTP activations had a mean injury severity score of 32. Blood use averaged 6.6 units of red blood cells (RBCs), 6.5 units of plasma, and 1.2 units of apheresis platelets. Fourteen activations ended without the administration of any blood products, and 45 (14%) did not meet the critical administration threshold of three components. Only 60 (19%) activations met the historic definition of massive with at least 10 units of RBCs administered. Mortality was 15% for the trauma-related activations. CONCLUSIONS: Massive transfusion protocol activations were frequent and conducted with high fidelity to the 1:1:1 unit ratio standard. Making blood components available quickly was associated with low rates of total component usage and low mortality for trauma patients and was not associated with overuse.

Building a New Transfusion Service.

OBJECTIVES: For over 60 years, Harborview Medical Center (HMC) in Seattle has received its blood components and pretransfusion testing from a centralized transfusion service operated by the regional blood supplier. In 2011, a hospital-based transfusion service (HBTS) was activated. METHODS: After 5 years of operation, we evaluated the effects of the HBTS by reviewing records of hospital blood use, quality system events, blood product delivery times, and costs. Furthermore, the effects of in-house expertise on laboratory medicine resident and medical laboratory scientist student training, as well as regulatory and accrediting agency concerns, were reviewed. RESULTS: Blood use records from 2003 to 2015 demonstrated large reductions in blood component procurement, allocation, transfusion, and wastage with decreases in costs temporally related to the change in service. The turnaround time for thawed plasma for trauma patients decreased from 90 to 3 minutes. Transfusion medicine education metrics for residents and laboratory technology students improved significantly. HMC researchers brought in $2 million in transfusion research funding. CONCLUSIONS: HMC successfully transitioned to an HBTS, providing world-class primary transfusion support to a level 1 trauma center. Near-term benefits in patient care, education, and research resulted. Blood support became faster, safer, and cheaper.

Optimization of stability, encapsulation, release, and cross-priming of tumor antigen-containing PLGA nanoparticles.

PURPOSE: In order to investigate Poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NP) as potential vehicles for efficient tumor antigen (TA) delivery to dendritic cells (DC), this study aimed to optimize encapsulation/release kinetics before determining immunogenicity of antigen-containing NP. METHODS: Various techniques were used to liberate TA from cell lines. Single (gp100) and multiple (B16-tumor lysate containing gp100) antigens were encapsulated within differing molecular weight PLGA co-polymers. Differences in morphology, encapsulation/release and biologic potency were studied. Findings were adopted to encapsulate fresh tumor lysate from patients with advanced tumors and compare stimulation of tumor infiltrating lymphocytes (TIL) against that achieved by soluble lysate. RESULTS: Four cycles of freeze-thaw + 15 s sonication resulted in antigen-rich lysates without the need for toxic detergents or protease inhibitors. The 80 KDa polymer resulted in maximal release of payload and favorable production of immunostimulatory IL-2 and IFN-γ. NP-mediated antigen delivery led to increased IFN-γ and decreased immunoinhibitory IL-10 synthesis when compared to soluble lysate. CONCLUSIONS: Four cycles of freeze-thaw followed by 15 s sonication is the ideal technique to obtain complex TA for encapsulation. The 80 KDa polymer has the most promising combination of release kinetics and biologic potency. Encapsulated antigens are immunogenic and evoke favorable TIL-mediated anti-tumor responses.

Polymer nanoparticles containing tumor lysates as antigen delivery vehicles for dendritic cell-based antitumor immunotherapy.

Encapsulation of tumor-associated antigens in polymer nanoparticles (NP) is a promising approach to enhance efficiency of antigen delivery for anti-tumor vaccines. Head and neck squamous carcinoma (HNSCC) cell lines were initially used to generate tumor-associated antigens (TAA)-containing poly (lactic-co-glycolic acid) (PLGA) NP; encapsulation efficiency and release kinetics were profiled. Findings were adopted to entrap fresh tumor lysate from five patients with advanced HNSCC. To test the hypothesis that NP enhance antigen presentation, dendritic cell (DC) produced from patient blood monocyte precursors were loaded with either the un-encapsulated or NP-encapsulated versions of tumor lysates. These were used to stimulate freshly-isolated autologous CD8+ T cells. In four of five patients, anti-tumor CD8+ T cells showed significantly increased immunostimulatory IFN-γ (p=0.071) or decreased immmunoinhibitory IL-10 production (p=0.0004) associated with NP-mediated antigen delivery. The observations represent an enabling step in the production of clinically-translatable, inexpensive, highly-efficient, and personalized polymer-based immunotherapy for solid organ malignancies. FROM THE CLINICAL EDITOR: Enhancing the antigen presentation may be a viable approach to increase the efficiency of tumor cell directed cytotoxicity via immune mechanisms. This study presents an example for this using head and neck cancer cell lines and nanotechnology-based encapsulated antigen presentation to dendritic cells. The observed CD8+ T-cell response was significantly enhanced. This method may pave the way to a highly efficient cancer cell elimination method with minimal to no toxicity.

Enhanced and prolonged cross-presentation following endosomal escape of exogenous antigens encapsulated in biodegradable nanoparticles.

CD8(+) T-cell responses are critical in the immunological control of tumours and infectious diseases. To prime CD8(+) T cells against these cell-associated antigens, exogenous antigens must be cross-presented by professional antigen-presenting cells (APCs). While cross-presentation of soluble antigens by dendritic cells is detectable in vivo, the efficiency is low, limiting the clinical utility of protein-based vaccinations. To enhance the efficiency of presentation, we generated nanoparticles from a biodegradable polymer, poly(D,L-lactide-co-glycolide) (PLGA), to deliver antigen into the major histocompatibility complex (MHC) class I antigen presentation pathway. In primary mouse bone marrow-derived dendritic cells (BMDCs), the MHC class I presentation of PLGA-encapsulated ovalbumin (OVA) stimulated T cell interleukin-2 secretion at 1000-fold lower concentration than soluble antigen and 10-fold lower than antigen-coated latex beads. The microparticles also served as an intracellular antigen reservoir, leading to sustained MHC class I presentation of OVA for 72 hr, decreasing by only 20% after 96 hr, a time at which the presentation of soluble and latex bead-associated antigens was undetectable. Cytosol extraction demonstrated that antigen delivery via PLGA particles increased the amount of protein that escaped from endosomes into the cytoplasm, thereby increasing the access of exogenous antigen to the classic MHC class I loading pathway. These data indicate that the unique properties of PLGA particle-mediated antigen delivery dramatically enhance and sustain exogenous antigen presentation by MHC class I, potentially facilitating the clinical use of these particles in vaccination.

Generation of dendritic cells from rabbit bone marrow mononuclear cell cultures supplemented with hGM-CSF and hIL-4.

The in vitro generation of dendritic cells (DCs) from either blood or bone marrow has been accomplished for humans and a number of other species. This ability has facilitated the opportunity to test the efficacy of DC vaccines in various tumor models. The cottontail rabbit papillomavirus (CRPV) model is the most clinically relevant animal model for human papillomavirus (HPV)-associated carcinogenesis. The CRPV model has been used to test various preventative and therapeutic vaccination strategies, and the availability of rabbit DCs would further expand its utility. However, to date, rabbit DCs have not been phenotypically and/or functionally characterized. Here we show that DCs can be generated in vitro from rabbit bone marrow mononuclear cells (BMMCs) cultured in the presence of the human cytokines GM-CSF and IL-4 and matured with lipopolysaccharide (LPS). These cells show upregulation of MHC class II and CD86, as well as downregulation of CD14, do not have non-specific esterase activity, are able to perform receptor-mediated endocytosis, and are potent stimulators of allogeneic T cell proliferation in mixed lymphocyte reactions. The ability to generate rabbit DCs makes it possible to test the efficacy of DC vaccination in the prevention and treatment of CRPV-induced lesions, which may provide useful preclinical data regarding the use of DC vaccines for HPV-associated lesions, including cervical cancer.