The use of mesenchymal stromal cell secretome to enhance guided bone regeneration in comparison with leukocyte and platelet-rich fibrin.

OBJECTIVES: Secretomes of mesenchymal stromal cells (MSC) represent a novel strategy for growth-factor delivery for tissue regeneration. The objective of this study was to compare the efficacy of adjunctive use of conditioned media of bone-marrow MSC (MSC-CM) with collagen barrier membranes vs. adjunctive use of conditioned media of leukocyte- and platelet-rich fibrin (PRF-CM), a current growth-factor therapy, for guided bone regeneration (GBR). METHODS: MSC-CM and PRF-CM prepared from healthy human donors were subjected to proteomic analysis using mass spectrometry and multiplex immunoassay. Collagen membranes functionalized with MSC-CM or PRF-CM were applied on critical-size rat calvaria defects and new bone formation was assessed via three-dimensional (3D) micro-CT analysis of total defect volume (2 and 4 weeks) and 2D histomorphometric analysis of central defect regions (4 weeks). RESULTS: While both MSC-CM and PRF-CM revealed several bone-related proteins, differentially expressed proteins, especially extracellular matrix components, were increased in MSC-CM. In rat calvaria defects, micro-CT revealed greater total bone coverage in the MSC-CM group after 2 and 4 weeks. Histologically, both groups showed a combination of regular new bone and 'hybrid' new bone, which was formed within the membrane compartment and characterized by incorporation of mineralized collagen fibers. Histomorphometry in central defect sections revealed greater hybrid bone area in the MSC-CM group, while the total new bone area was similar between groups. CONCLUSION: Based on the in vitro and in vivo investigations herein, functionalization of membranes with MSC-CM represents a promising strategy to enhance GBR.

Proteomic Analysis of Mesenchymal Stromal Cells Secretome in Comparison to Leukocyte- and Platelet-Rich Fibrin.

Secretomes of mesenchymal stromal cells (MSCs) are emerging as a novel growth factor (GF)-based strategy for periodontal and bone regeneration. The objective of this study was to compare the secretome of human bone marrow MSC (BMSC) to that of leukocyte- and platelet-rich fibrin (L-PRF), an established GF-based therapy, in the context of wound healing and regeneration. Conditioned media from human BMSCs (BMSC-CM) and L-PRF (LPRF-CM) were subjected to quantitative proteomic analysis using liquid chromatography with tandem mass spectrometry. Global profiles, gene ontology (GO) categories, differentially expressed proteins (DEPs), and gene set enrichment (GSEA) were identified using bioinformatic methods. Concentrations of selected proteins were determined using a multiplex immunoassay. Among the proteins identified in BMSC-CM (2157 proteins) and LPRF-CM (1420 proteins), 1283 proteins were common. GO analysis revealed similarities between the groups in terms of biological processes (cellular organization, protein metabolism) and molecular functions (cellular/protein-binding). Notably, more DEPs were identified in BMSC-CM (n = 550) compared to LPRF-CM (n = 118); these included several key GF, cytokines, and extracellular matrix (ECM) proteins involved in wound healing. GSEA revealed enrichment of ECM (especially bone ECM)-related processes in BMSC-CM and immune-related processes in LPRF-CM. Similar trends for intergroup differences in protein detection were observed in the multiplex analysis. Thus, the secretome of BMSC is enriched for proteins/processes relevant for periodontal and bone regeneration. The in vivo efficacy of this therapy should be evaluated in future studies.

COVID-19 patients treated with convalescent plasma. / Covid-19-pasienter behandlet med rekonvalesensplasma.

BACKGROUND: In Norway, treatment with COVID-19 convalescent plasma has been given through the NORPLASMA project. The treatment was initially offered to critically ill patients after an individual assessment, but from December 2020, the indication was limited to critically ill, immunocompromised patients. In this article we describe clinical characteristics, comorbidity and mortality in patients who received convalescent plasma in these two periods. MATERIAL AND METHOD: From 22 April 2020 to 30 March 2022, a total of 79 patients were included in the observational studies NORPLASMA MONITOR and the Norwegian SARS-CoV-2 study. The patients had received a total of 193 units of convalescent plasma at 15 Norwegian hospitals/nursing homes; 62 in South-Eastern Norway Regional Health Authority, 8 in Western Norway Regional Health Authority and 9 in Central Norway Regional Health Authority. Information on immune status, comorbidity and course of infection was retrieved from the patient records after informed written consent was obtained. RESULTS: Of 79 patients with a median age of 65 years (interquartile range 51-⁠73) who were treated with convalescent plasma, 31 (39 %) died during hospitalisation. A total of 59 patients were immunocompromised, and of these, 20 died in hospital compared to 11 of 20 who were assumed to be immunocompetent. Median number of comorbidities was 2 (interquartile range 1-4). The patients received a median of two plasma units (min.-max. 1-21). Two of the patients developed mild allergic skin reactions. INTERPRETATION: Convalescent plasma was well tolerated by patients with COVID-19. Immunocompromised patients may have benefitted from the treatment, with lower mortality than for those assumed to be immunocompetent.

Convalescent plasma from Norwegian blood donors to treat COVID-19. / Rekonvalesensplasma mot covid-19 fremstilt fra norske blodgivere.

BACKGROUND: At the start of the pandemic, the Norwegian Directorate of Health and Norwegian blood banks initiated the production of COVID-19 convalescent plasma within the framework of clinical studies. In this article we describe the blood donors who participated. MATERIAL AND METHOD: Blood donors who had recovered from COVID-19 were recruited to donate single donor plasma for the purpose of patient treatment. Data on the course of infection, leukocyte antibodies and antibody level against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) per plasma unit were registered after informed consent was obtained. We calculated a disease score defined as the total number of self-reported symptoms/findings and hospitalisation where relevant (score 0-⁠11). RESULTS: A total of 1644 plasma units were collected from 266 plasma donors at 12 blood banks. Median disease score was 5 (interquartile range 3-⁠6), and 15 donors had recovered from pneumonia and/or been hospitalised. A total of 599/1644 plasma units from 106/266 donors met our requirement for SARS-CoV-2 antibody content (> 60 % inhibition of virus binding to angiotensin-converting enzyme 2 (ACE2)) or positive virus neutralisation test. The antibody level in donors waned over time following infection, and showed no clear correlation with disease score. INTERPRETATION: The number of symptoms and findings in blood donors could not predict antibody response at individual level, and antibody testing was crucial for the production of effective convalescent plasma.

Training of medical students in the use of emergency whole blood collection and transfusion in the framework of a civilian walking blood.

INTRODUCTION: In this report, we describe a training program in emergency whole blood collection and transfusion for medical students at the University of Bergen. The overall aim of the program is to improve the availability of early balanced blood transfusion for the treatment of patients with life-threatening bleeding in rural health care services. STUDY DESIGN AND METHODS: The voluntary training program provides the knowledge needed to practice emergency whole blood transfusions and understand the system for emergency whole blood collection in the framework of a civilian walking blood bank (WBB). It includes theoretical and practical sessions. In-person teaching and web-based learning resources are provided. An anonymous survey of the students attending the training course in the autumn of 2022 and spring 2023 was performed. RESULTS: 128 of 178 students participated in the practical training. 88 of 128 (69%) responded to the survey. 82 (93%) performed blood typing, 71 (81%) performed donor interviews, 61 (69%) partially performed whole blood collection (up to blood in bag) and 27 (30%) participated in complete whole blood collection and performed autologous reinfusion. No complications occurred during training. The students reported that the training course increased their understanding of how to ensure access to emergency blood transfusion by the use of a WBB. DISCUSSION: Structured theoretical and practical training in emergency whole blood collection and emergency transfusion is feasible and of interest to medical students. A multidisciplinary approach to student training in emergency whole blood collection and transfusion should be considered.

Functionalizing Collagen Membranes with MSC-Conditioned Media Promotes Guided Bone Regeneration in Rat Calvarial Defects.

Functionalizing biomaterials with conditioned media (CM) from mesenchymal stromal cells (MSC) is a promising strategy for enhancing the outcomes of guided bone regeneration (GBR). This study aimed to evaluate the bone regenerative potential of collagen membranes (MEM) functionalized with CM from human bone marrow MSC (MEM-CM) in critical size rat calvarial defects. MEM-CM prepared via soaking (CM-SOAK) or soaking followed by lyophilization (CM-LYO) were applied to critical size rat calvarial defects. Control treatments included native MEM, MEM with rat MSC (CEL) and no treatment. New bone formation was analyzed via micro-CT (2 and 4 weeks) and histology (4 weeks). Greater radiographic new bone formation occurred at 2 weeks in the CM-LYO group vs. all other groups. After 4 weeks, only the CM-LYO group was superior to the untreated control group, whereas the CM-SOAK, CEL and native MEM groups were similar. Histologically, the regenerated tissues showed a combination of regular new bone and hybrid new bone, which formed within the membrane compartment and was characterized by the incorporation of mineralized MEM fibers. Areas of new bone formation and MEM mineralization were greatest in the CM-LYO group. Proteomic analysis of lyophilized CM revealed the enrichment of several proteins and biological processes related to bone formation. In summary, lyophilized MEM-CM enhanced new bone formation in rat calvarial defects, thus representing a novel 'off-the-shelf' strategy for GBR.

Implementation of a low-titre whole blood transfusion program in a civilian helicopter emergency medical service.

BACKGROUND: Early balanced transfusion is associated with improved outcome in haemorrhagic shock patients. This study describes the implementation and evaluates the safety of a whole blood transfusion program in a civilian helicopter emergency medical service (HEMS). METHODS: This prospective observational study was performed over a 5-year period at HEMS-Bergen, Norway. Patients in haemorrhagic shock receiving out of hospital transfusion of low-titre Group O whole blood (LTOWB) or other blood components were included. Two LTOWB units were produced weekly and rotated to the HEMS for forward storage. The primary endpoints were the number of patients transfused, mechanisms of injury/illness, adverse events and survival rates. Informed consent covered patient pathway from time of emergency interventions to last endpoint and subsequent data handling/storage. RESULTS: The HEMS responded to 5124 patients. Seventy-two (1.4%) patients received transfusions. Twenty patients (28%) were excluded due to lack of consent (16) or not meeting the inclusion criteria (4). Of the 52 (100%) patients, 48 (92%) received LTOWB, nine (17%) received packed red blood cells (PRBC), and nine (17%) received freeze-dried plasma. Of the forty-six (88%) patients admitted alive to hospital, 35 (76%) received additional blood transfusions during the first 24 h. Categories were blunt trauma 30 (58%), penetrating trauma 7 (13%), and nontrauma 15 (29%). The majority (79%) were male, with a median age of 49 (IQR 27-70) years. No transfusion reactions, serious complications or logistical challenges were reported. Overall, 36 (69%) patients survived 24 h, and 28 (54%) survived 30 days. CONCLUSIONS: Implementing a whole blood transfusion program in civilian HEMS is feasible and safe and the logistics around out of hospital whole blood transfusions are manageable. Trial registration The study is registered in the ClinicalTrials.gov registry (NCT02784951).

Brief communication: Effects of conditioned media from human platelet lysate cultured MSC on osteogenic cell differentiation in vitro.

Culturing mesenchymal stromal cells (MSC) in human platelet lysate (HPL) supplemented media can enhance their osteogenic differentiation potential. The objective of this study was to test the hypothesis that conditioned media (CM) derived from HPL-cultured MSC also have pro-osteogenic effects. Pooled CM was prepared from HPL-cultured human bone marrow MSC (BMSC) of multiple donors and applied on BMSC of different donors (than those used for CM preparation), with or without additional supplementation [HPL, fetal bovine serum (FBS)] and osteogenic stimulation. At various time-points, cell proliferation, alkaline phosphatase (ALP) activity, osteogenic gene expression and in vitro mineralization were assessed. BMSC in standard unstimulated growth media served as controls. After 3-7 days, CM alone did not promote BMSC proliferation or ALP activity; supplementation of CM with HPL slightly improved these effects. After 2 and 7 days, CM alone, but not CM supplemented with HPL, promoted osteogenic gene expression. After 14 days, only CM supplemented with FBS and osteogenic stimulants supported in vitro BMSC mineralization; CM alone and CM supplemented with HPL did not support mineralization, regardless of osteogenic stimulation. In summary, CM from HPL-cultured BMSC promoted osteogenic gene expression but not in vitro mineralization in allogeneic BMSC even when supplemented with HPL and/or osteogenic stimulants. Future studies should investigate the role and relevance of supplementation and osteogenic induction in in vitro assays using CM from MSC.

Implementation of a dual platelet inventory in a tertiary hospital during the COVID-19 pandemic enabling cold-stored apheresis platelets for treatment of actively bleeding patients.

BACKGROUND: To increase preparedness and mitigate the risk of platelet shortage without increasing the number of collections, we introduced a dual platelet inventory with cold-stored platelets (CSP) with 14-days shelf life for actively bleeding patients during the COVID-19 pandemic. STUDY DESIGN AND METHODS: We collected apheresis platelet concentrates with blood type O or A. All patients receiving CSP units were included in a quality registry. Efficacy was evaluated by total blood usage and laboratory analysis of platelet count, hemoglobin, and TEG 6s global hemostasis assay. Feasibility was evaluated by monitoring inventory and a survey among laboratory staff. RESULTS: From 17 March, 2020, to 31 December, 2021, we produced 276 CSP units and transfused 186 units to 92 patients. Main indication for transfusion was surgical bleeding (88%). No transfusion reactions were reported. 24-h post-transfusion patient survival was 96%. Total outdate in the study period was 33%. The majority (75%) of survey respondents answered that they had received sufficient information and training before CSP was implemented. Lack of information about bleeding status while issuing platelets, high workload, and separate storage location was described as main reasons for outdates. DISCUSSION: CSP with 14-days shelf life is a feasible alternative for the treatment of patients with bleeding. Implementation of a dual platelet inventory requires thorough planning, including information and training of clinical and laboratory staff, continuous follow-up of practice and patients, and an easy-to-follow algorithm for use of CSP units. A dual platelet inventory may mitigate the risk of platelet shortage during a pandemic situation.

In vitro quality and hemostatic function of cold-stored CPDA-1 whole blood after repeated transient exposure to 28°C storage temperature.

BACKGROUND: Blood products are frequently exposed to room temperature or higher for longer periods than permitted by policy. We aimed to investigate if this resulted in a measurable effect on common quality parameters and viscoelastic hemostatic function of cold stored CPDA-1 whole blood. STUDY DESIGN AND METHODS: 450 ml of whole blood from 16 O Rh(D) positive donors was collected in 63 ml of CPDA-1 and stored cold. Eights bags were exposed to five weekly 4-h long transient temperature changes to 28°C. Eight bags were stored continuously at 4°C as a control. Samples were collected at baseline on day 1, after the first cycle on day 1 and weekly before each subsequent cycle (day 7, 14, 21, 28 and 35). Hemolysis, hematological parameters, pH, glucose, lactate, potassium, thromboelastography, INR, APTT, fibrinogen, and factor VIII were measured. RESULTS: CPDA-1 whole blood repeatedly exposed to 28°C did not show reduced quality compared to the control group on day 35. Two units in the test group had hemolysis of 1.1% and 1.2%, and two in the control group hemolysis of 0.8%. Remaining thromboelastography clot strength (MA) on day 35 was 51.7 mm (44.8, 58.6) in the test group and 46.1 (41.6, 50.6) in the control group (p = .023). Platelet count was better preserved in the test group (166.7 [137.8, 195.6] vs. 117.8 [90.3, 145.2], p = .018). One sample in the test group was positive for Cutibacterium acnes on day 35 + 6. CONCLUSION: Hemolysis findings warrant further investigation. Other indicators of quality were not negatively affected.

A patient with severe COVID-19 treated with convalescent plasma. / Pasient med alvorlig covid-19 behandlet med rekonvalesensplasma.

BACKGROUND: COVID-19 can lead to life-threatening disease. While awaiting vaccines or documented specific therapeutic agents, several alternative treatment options are under investigation. This is a case report of the first COVID-19 patient treated with convalescent plasma in Norway. CASE PRESENTATION: A patient with severe COVID-19 on prolonged mechanical ventilation, who was PCR SARS-Cov-2 positive on day 22, was transfused with convalescent plasma on day 31 and tested negative for SARS-CoV-2 the following day. The patient gradually improved and was weaned from the ventilator and discharged alive from the ICU on day 63. INTERPRETATION: This case report concerns one patient with clinical improvement after convalescent plasma transfusion. A SARS-CoV-2 test was not performed immediately before transfusion and the complexity of intensive care treatment makes it difficult to draw any conclusions on the potential effectiveness of this treatment. However, this case report is encouraging with regard to planned trials with convalescent plasma.

How do I implement a whole blood-based blood preparedness program in a small rural hospital?

Civilian and military guidelines recommend balanced transfusion to patients with life-threatening bleeding. Early start of transfusion has shown improved survival. Thus, a balanced blood inventory must be available in all levels of health care to ensure early stabilization and damage control resuscitation of patients with bleeding. Whole blood has been reintroduced as a blood product for massive bleeding situations because it affords plasma, red blood cells, and platelets in a balanced ratio in a logistically advantageous way. In this article, we describe how to establish a whole blood-based blood preparedness program in a small rural hospital with limited resources. We present an implementation tool kit, which includes discussions on whole blood program strategies and the process of developing detailed procedures on donor selection, collection, storage, and transfusion management of whole blood. The importance of training and audit of the routines is highlighted, and establishment of an emergency walking blood bank is discussed. We conclude that implementation of a whole blood program is achievable in small rural hospitals and recommend that rural health care facilities at all treatment levels enable early balanced transfusion for patients with life-threatening bleeding by establishing protocols for whole blood-based preparedness.

Cold-stored whole blood in a Norwegian emergency helicopter service: an observational study on storage conditions and product quality.

BACKGROUND: Increasing numbers of emergency medical service agencies and hospitals are developing the capability to administer blood products to patients with hemorrhagic shock. Cold-stored whole blood (WB) is the only single product available to prehospital providers who aim to deliver a balanced resuscitation strategy. However, there are no data on the safety and in vitro characteristics of prehospital stored WB. This study aimed to describe the effects on in vitro quality of storing WB at remote helicopter bases in thermal insulating containers. STUDY DESIGN AND METHODS: We conducted a two-armed single-center study. Twenty units (test) were stored in airtight thermal insulating containers, and 20 units (controls) were stored according to routine procedures in the Haukeland University Hospital Blood Bank. Storage conditions were continuously monitored during emergency medical services missions and throughout remote and blood bank storage. Hematologic and metabolic variables, viscoelastic properties, and platelet (PLT) aggregation were measured on Days 1, 8, 14, and 21. RESULTS: Storage conditions complied with the EU guidelines throughout remote and in-hospital storage for 21 days. There were no significant differences in PLT aggregation, viscoelastic properties, and hematology variables between the two groups. Minor significantly lower pH, glucose, and base excess and higher lactate were observed after storage in airtight containers. CONCLUSION: Forward cold storage of WB is safe and complies with EU standards. No difference is observed in hemostatic properties. Minor differences in metabolic variables may be related to the anaerobic conditions within the thermal box.

Cold-stored leukoreduced CPDA-1 whole blood: in vitro quality and hemostatic properties.

BACKGROUND: Some jurisdictions require leukoreduction of cellular blood components. The only whole blood collection set with a platelet-saving filter uses citrate-phosphate-dextrose (CPD) as storage solution. Substituting CPD with citrate-phosphate-dextrose-adenine (CPDA-1) increases shelf life from 21 to 35 days. This would simplify prehospital and rural resupply and reduce wastage. We investigated in vitro quality and hemostatic properties of CPDA-1 whole blood leukoreduced with a platelet-saving filter. STUDY DESIGN AND METHODS: CPDA-1 whole blood was leukoreduced using a platelet-saving filter and stored 35 days. EDQM requirements, hematology, metabolic parameters, thromboelastography, light transmission aggregometry, fibrinogen, factor VIII, and interleukin-6 were measured on Days 0, 1, 14, 21, and 35 and compared to non-leukoreduced blood. RESULTS: All units met EDQM requirements. Leukoreduction yielded residual white blood cell count <1 × 106 and 87% platelet recovery on Day 1. It caused reduction in thromboelastography parameters, but not aggregometry response. No hemolysis >0.8% was observed. Factor VIII was higher on Day 35 in the leukoreduced group, 37.9 (95% CI: 26.0, 49.8) versus 13.8 (9.4, 18.2) IU/dL. In both groups, aggregation was significantly reduced by Day 14. Thromboelastography showed remaining platelet activity on Day 35, MA 46.9 (42.1, 51.7) in the leukoreduced and 44.3 (39.6, 49.0) mm in the non-leukoreduced group. Fibrinogen was within reference ranges at Day 35 (>2 g/dL). Interleukin-6 was not detectable. CONCLUSION: Leukoreducing CPDA-1 whole blood with a platelet-saving filter did not compromise hemostatic properties. We encourage development of a single bag CPDA-1 whole blood collection set with in-line platelet-saving filter.

Environmental pollutants in blood donors: The multicentre Norwegian donor study.

OBJECTIVES: The aim of this study was to measure blood concentrations of environmental pollutants in Norwegian donors and evaluate the risk of pollutant exposure through blood transfusions. BACKGROUND: Transfused blood may be a potential source of exposure to heavy metals and organic pollutants and presents a risk to vulnerable patient groups such as premature infants. METHODS/MATERIALS: Donors were randomly recruited from three Norwegian blood banks: in Bergen, Tromsø and Kirkenes. Selected heavy metals were measured in whole blood using inductively coupled plasma mass spectrometry (ICP-MS), and perfluoroalkyl substances (PFAS) were measured in serum by ultrahigh-pressure liquid chromatography coupled with a triple-quadrupole mass spectrometer (UHPLC-MS/MS). RESULTS: Almost 18% of blood donors had lead concentrations over the limit suggested for transfusions in premature infants (0.09 µmol/L). About 11% of all donors had mercury concentrations over the suggested limit of 23.7 nmol/L. Cadmium was higher than the limit, 16 nmol/L, in 4% of donors. Perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS) concentrations were over the suggested limit of 0.91 ng/mL in 68% and 100% of the donors, respectively. PFAS concentrations and heavy metal concentrations increased with donor's age. CONCLUSION: A considerable percentage of donors had lead, PFOS and PFOA concentrations over the suggested limits. In addition, at each study site, there were donors with high mercury and cadmium concentrations. Selecting young donors for transfusions or measurements of pollutants in donor blood may be a feasible approach to avoid exposure through blood transfusions to vulnerable groups of patients such as premature infants.

Antibodies to Multiple Receptors are Associated with Neuropsychiatric Symptoms and Mortality in Alzheimer's Disease: A Longitudinal Study.

BACKGROUND: Endogenous antibodies to signaling molecules and receptors (Abs) are associated with Alzheimer's disease (AD). OBJECTIVES: To investigate the association of 33 Abs to dopaminergic, serotoninergic, muscarinic, adrenergic, vascular, and immune receptors with cognitive, neuropsychiatric, and mortality outcomes. METHODS: Ninety-one patients with mild AD were followed annually for 5 years with the Mini-Mental State Examination (MMSE) and the Neuropsychiatric Inventory (NPI; composite outcomes: "psychosis" (item 1 + 2), "mood" (item 4 + 5 + 7), and "agitation" (item 3 + 8 + 9)). Abs were quantified in sera obtained at baseline by ELISA and reduced to principal components (PCs). Associations between Abs and outcomes were assessed by a mixed model (MMSE decline), zero-inflated fixed effects count models (composite NPI scores), and Cox regression (mortality). The resulting p-values were adjusted for multiple testing according to a false discovery rate of 0.05 (Benjamini-Hochberg). RESULTS: The measured levels of the 33 Abs formed four PCs. PC1 (dopaminergic and serotonergic Abs) was associated with increased mortality (Hazard ratio 2.57, p < 0.001), PC2 (serotonergic, immune, and vascular Abs) with decreased agitation symptoms (ß - 0.19, p < 0.001), and PC3 (cholinergic receptor Abs) with increased mood symptoms (ß 0.04, p = 0.002), over time. There were no associations between Abs and MMSE decline. CONCLUSION: The associations between Abs, mortality, and neuropsychiatric symptoms reported in this cohort are intriguing. They cannot, however, be generalized. Validation in independent sample sets is required.

Peripheral Blood Stem Cell Mobilization in Healthy Donors by Granulocyte Colony-Stimulating Factor Causes Preferential Mobilization of Lymphocyte Subsets.

Background: Allogeneic hematopoietic stem cell transplantation is associated with a high risk of immune-mediated post-transplant complications. Graft depletion of immunocompetent cell subsets is regarded as a possible strategy to reduce this risk without reducing antileukemic immune reactivity. Study design and methods: We investigated the effect of hematopoietic stem cell mobilization with granulocyte colony-stimulating factor (G-CSF) on peripheral blood and stem cell graft levels of various T, B, and NK cell subsets in healthy donors. The results from flow cytometric cell quantification were examined by bioinformatics analyses. Results: The G-CSF-induced mobilization of lymphocytes was a non-random process with preferential mobilization of naïve CD4+ and CD8+ T cells together with T cell receptor αß+ T cells, naïve T regulatory cells, type 1 T regulatory cells, mature and memory B cells, and cytokine-producing NK cells. Analysis of circulating lymphoid cell capacity to release various cytokines (IFNγ, IL10, TGFß, IL4, IL9, IL17, and IL22) showed preferential mobilization of IL10 releasing CD4+ T cells and CD3-19- cells. During G-CSF treatment, the healthy donors formed two subsets with generally strong and weaker mobilization of immunocompetent cells, respectively; hence the donors differed in their G-CSF responsiveness with regard to mobilization of immunocompetent cells. The different responsiveness was not reflected in the graft levels of various immunocompetent cell subsets. Furthermore, differences in donor G-CSF responsiveness were associated with time until platelet engraftment. Finally, strong G-CSF-induced mobilization of various T cell subsets seemed to increase the risk of recipient acute graft versus host disease, and this was independent of the graft T cell levels. Conclusion: Healthy donors differ in their G-CSF responsiveness and preferential mobilization of immunocompetent cells. This difference seems to influence post-transplant recipient outcomes.

Novel Nanoparticulate and Ionic Titanium Antigens for Hypersensitivity Testing.

Titanium is used in a wide variety of materials ranging from medical devices to materials used in everyday life. Adverse biological reactions that could occur in patients, consumers, and workers should be monitored and prevented. There is a lack of available agents to test and predict titanium-related hypersensitivity. The aim of this study was to develop two bioavailable titanium substances in ionic and nanoparticulate form to serve as antigens for hypersensitivity testing in vitro. Peripheral blood mononuclear cells from 20 test subjects were stimulated with the antigens and secretion of monocytic and lymphatic cytokines and chemokines were measured by a multiplex bead assay. Lymphocyte stimulation indices were also determined in a subset of test subjects by measuring CD69 and HLA-DR expression by flow cytometry. Cytokine profiling revealed that both antigens increased production of typical monocyte and macrophage secreted cytokines after 24 h, with significant increases in IL-1ß, IL-7, IL-10, IL-12, IL-2R, IL-6, GM-CSF, TNF-α, IL-1RA, MIP-1α, MIP-1ß, IFN-α, and IL-15. Lymphatic cytokines and chemokines were not significantly induced by activation. After seven days of stimulation, ionic-Ti (2.5 µg/mL) caused proliferation (stimulation index > 2) of CD4+ cells and CD8+ cells in all persons tested (N = 6), while titanium dioxide nanoparticles (50 µg/mL) only caused significant proliferation of CD4+ cells. Our preliminary results show that the experimental titanium antigens, especially the ionic form, induce a general inflammatory response in vitro. A relevant cohort of test subjects is required to further elucidate their potential for predictive hypersensitivity testing.

Preparation of leukoreduced whole blood for transfusion in austere environments; effects of forced filtration, storage agitation, and high temperatures on hemostatic function.

BACKGROUND: Damage control resuscitation principles advocate the use of blood to treat traumatic hemorrhage. Hemorrhage is a leading cause of preventable death on the battlefield, but making blood components available far forward presents logistical challenges due to shelf life and storage requirements. Whole blood simplifies logistics and enables collection in the field but can cause leukocyte-related transfusion reactions. A field-adapted leukoreduction system must be fast and safe, and storage of whole blood should preserve hemostatic function. METHODS: Blood was collected using Imuflex WB-SP and leukoreduced at 0, 150, or 300 mm Hg. Additional bags were stored at 4°C for 21 days unagitated, mixed daily, agitated or head-over-heel rotated, at 22°C for 3 days, or 32°C for 2 hours. Hematology, coagulation, CD62P/CD42b, thromboelastography (TEG)/thromboelastometry (ROTEM), and Multiplate was performed. RESULTS: Filtration time was 35 ± 1, 14 ± 0, and 9 ± 0 minutes at 0, 150, and 300 mm Hg, respectively. One of 10 units at 150 mm Hg and 4 of 11 at 300 mm Hg had residual whole blood cells greater than 5.0 × 10 per unit. One of 11 at 300 mm Hg had platelet recovery of less than 80%. Hemolysis was less than 0.2%. Filtration decreased thromboelastography/thromboelastometry and Multiplate aggregation response. Stored at 4°C, α and MA/MCF moderately decreased regardless of mixing. Significant loss of aggregation response and increased CD62P expression was seen by Day 10. By Day 3, storage at 22°C caused loss of most aggregation. Two-hour storage at 32°C did not significantly affect hemostatic capacity. CONCLUSION: Forced filtration reduced leukoreduction time, but increased residual whole blood cells reduced hemostatic function. Aggregation response deteriorated early in storage, while viscoelastic assays decreased more gradually. Mixing showed no benefits. LEVEL OF EVIDENCE: Diagnostic study, level IV.

The healthy donor profile of immunoregulatory soluble mediators is altered by stem cell mobilization and apheresis.

BACKGROUND: Peripheral blood stem cells from healthy donors mobilized by granulocyte colony-stimulating factor (G-CSF) and thereafter harvested by leukapheresis are commonly used for allogeneic stem cell transplantation. METHODS: Plasma levels of 38 soluble mediators (cytokines, soluble adhesion molecules, proteases, protease inhibitors) were analyzed in samples derived from healthy stem cell donors before G-CSF treatment and after 4 days, both immediately before and after leukapheresis. RESULTS: Donors could be classified into two main subsets based on their plasma mediator profile before G-CSF treatment. Seventeen of 36 detectable mediators were significantly altered by G-CSF; generally an increase in mediator levels was seen, including pro-inflammatory cytokines, soluble adhesion molecules and proteases. Several leukocyte- and platelet-released mediators were increased during apheresis. Both plasma and graft mediator profiles were thus altered and showed correlations to graft concentrations of leukocytes and platelets; these concentrations were influenced by the apheresis device used. Finally, the mediator profile of the allotransplant recipients was altered by graft infusion, and based on their day +1 post-transplantation plasma profile our recipients could be divided into two major subsets that differed in overall survival. DISCUSSION: G-CSF alters the short-term plasma mediator profile of healthy stem cell donors. These effects together with the leukocyte and platelet levels in the graft determine the mediator profile of the stem cell grafts. Graft infusion also alters the systemic mediator profile of the recipients, but further studies are required to clarify whether such graft-induced alterations have a prognostic impact.