Potential for prolongation of fibrinogen concentrates post-reconstitution.

BACKGROUND AND OBJECTIVES: Reconstituted fibrinogen concentrate is considered stable for 8-24 h based on product monographs. Given the long half-life of fibrinogen in vivo (3-4 days), we hypothesized that reconstituted sterile fibrinogen protein would remain stable longer than 8-24 h. Extending the expiry date for reconstituted fibrinogen concentrate could decrease wastage and facilitate reconstitution in advance to minimize turnaround times. We performed a pilot study to define the stability of reconstituted fibrinogen concentrates over time. MATERIALS AND METHODS: Reconstituted Fibryga® (Octapharma AG) from 64 vials was stored in the temperature-controlled refrigerator (4 °C) for up to 7 days with functional fibrinogen concentration measured serially using the automated Clauss method. The samples were frozen, then thawed and diluted with pooled normal plasma in order for them to be batch tested. RESULTS: Reconstituted fibrinogen samples stored in the refrigerator showed no significant reduction in functional fibrinogen concentration for the entire 7-day study period (p = 0.63). Duration of initial freezing had no detrimental effect on functional fibrinogen levels (p = 0.23). CONCLUSION: Fibryga® can be stored at 2-8 °C post-reconstitution for up to one week with no loss in functional fibrinogen activity based on Clauss fibrinogen assay. Further studies with other fibrinogen concentrate formulations and clinical in vivo studies may be warranted.

Prediction of massive transfusion with the Revised Assessment of Bleeding and Transfusion (RABT) score at Canadian level I trauma centers.

BACKGROUND: Early damage control resuscitation and massive transfusion (MT) protocol activations improve outcomes in trauma patients with hemorrhagic shock, where scores to guide MT prediction are used including: the Assessment of Blood Consumption (ABC), Shock Index (SI), and Revised Assessment of Bleeding and Transfusion (RABT) scores. Our aim was to validate the RABT score in patients from two level I trauma centers in Canada. METHODS: A retrospective review of adult patients meeting trauma team activation criteria receiving >1 unit of red blood cells (RBCs) within 24 h of admission, from 2015 to 2020, was conducted. A RABT score ≥ 2, ABC score ≥ 2, and Shock Index (SI) ≥ 1 was used to predict MT using both research (≥10 RBCs in 24 h) and clinical (≥3 RBCs in 3 h) definitions. Scores were assessed and compared using sensitivity, specificity, and the area under the receiver operating characteristic (AUROC). RESULTS: We analyzed 514 patients with a mean age of 44.4 (19.2) years and a median injury severity score of 29 [18-38]. For both MT definitions, the RABT score trended towards higher sensitivity and lower specificity compared to ABC score and SI. For both research and clinical definitions of MT, the AUROC for the RABT score was not significantly higher (Research - RABT: 0.673 [0.610-0.735], ABC: 0.642 [0.551-0.734], SI 0.691 [0.625-0.757]; Clinical - RABT: 0.653 [0.608-0.698], ABC: 0.646 [0.600-0.691], SI 0.610 [0.559-0.660]). CONCLUSION: The RABT score is a valid tool for predicting the need for MTPs, performing similarly with a trend towards higher sensitivity when compared to the ABC score and SI.

Should intraoperative cell-salvaged blood be used in patients with suspected or known malignancy?

PURPOSE: Intraoperative cell salvage (ICS) is used as an alternative to allogeneic blood transfusion in an attempt to avoid or minimize the risks associated with allogeneic blood. Intraoperative cell salvage is generally avoided in surgeries where malignancy is confirmed or suspected due to concern for potential metastasis or cancer recurrence. The application of post-processing methods for ICS is hypothesized to eliminate this potential risk. The purpose of this narrative review is to examine the in vitro experimental evidence as it pertains to the removal of tumour cells from ICS blood and to review the clinical studies where ICS blood has been used in patients with malignancy. SOURCE: A search of the English literature for relevant articles published from 1973 to 2012 was undertaken using MEDLINE and Cochrane databases. Bibliographies were cross-referenced to locate further studies. PRINCIPAL FINDINGS: Leukoreduction filters are an effective method for removal of malignant cells from ICS blood. Small non-randomized clinical studies to date do not show evidence of an increased rate of metastasis or cancer recurrence. Although a theoretical risk of disease recurrence persists, the decision to use autologous ICS blood must be weighed against the known risks of allogeneic blood transfusion. CONCLUSION: Transfusion of autologous blood harvested via ICS should be considered a viable option for reduction or avoidance of allogeneic product during many oncologic surgeries and may be a lifesaving option for those patients who refuse allogeneic blood products.

Nutrition in primary care: current practices, attitudes, and barriers.

OBJECTIVE: To investigate what role family physicians currently play in the management of patients with nutrition-related issues and whether implementation of current nutrition counseling guidelines is feasible in primary care practices. DESIGN: Mailed survey. SETTING: Family practice offices in British Columbia. PARTICIPANTS: A total of 451 Canadian-trained family physicians practising in British Columbia. MAIN OUTCOME MEASURES: Respondents' demographic characteristics; respondents' attitudes about and perceived barriers to nutrition counseling, as well as their current practices and training in this area. RESULTS: Among the 757 physicians surveyed, the response rate was 59.6%. Overall, respondents had positive attitudes about the role of nutrition in patient health, and most physicians (58.1%) believed that more than 60% of their patients would benefit from nutrition counseling. However, there was a considerable gap between the proportion of patients who respondents thought would benefit from nutrition counseling and the proportion of patients who received such counseling either in the family physicians' offices or through referral to dietitians. Rural physicians referred patients to dietitian services more frequently than urban physicians did (41.7% vs 21.7% made more than 20 referrals to dietitians each year). Nearly all physicians identified lack of time and compensation as the strongest barriers to providing nutrition guidance. Training was not considered to be as strong a barrier to counseling, even though 82.3% of family physicians reported their formal nutrition training in medical school to be inadequate, and only 30% of family physicians reported currently using any nutrition-related resources. CONCLUSION: For family physicians, successful implementation of the 2006 Canadian Clinical Practice Guidelines on the Management and Prevention of Obesity requires access to adequate training, compensation, and evidence-based interventions related to nutrition. This study highlights current nutrition counseling practices in family medicine and identifies several obstacles to integrating the current guidelines in primary care settings.

A prospective audit of postoperative pain control in pediatric patients.

Pain is a subjective experience that is affected by physical, emotional, and psychological factors, and reliable assessment of pain can be a challenge in the pediatric population. A quality improvement project was conducted at one Canadian health care facility to examine the effectiveness of the postoperative pain management strategy for children admitted to the postanesthesia care unit (PACU). Effective control of postoperative pain involves several preventive strategies that include preoperative analgesia, appropriate use of intraoperative analgesic techniques, and identification of children at risk for significant postoperative pain. Successful implementation of these techniques requires a multidisciplinary team approach involving the patient, the PACU nurses, the anesthesia care provider, and other surgical team members.

In vivo control of acute lymphoblastic leukemia by immunostimulatory CpG oligonucleotides.

Despite considerable success in treating newly diagnosed childhood acute lymphoblastic leukemia (ALL), relapsed disease remains a significant clinical challenge. Using a NOD/SCID mouse xenograft model, we report that immunostimulatory DNA oligonucleotides containing CpG motifs (CpG ODNs) stimulate significant immune activity against primary human ALL cells in vivo. The administration of CpG ODNs induced a significant reduction in systemic leukemia burden, mediated continued disease control, and significantly improved survival of mice with established human ALL. The death of leukemia cells in vivo was independent of the ability of ALL cells to respond directly to CpG ODNs and correlated with the production of IL-12p70, IFN-alpha, and IFN-gamma by the host. In addition, depletion of natural killer cells by anti-asialo-GM1 treatment significantly reduced the in vivo antileukemic activity of CpG ODN. This antileukemia effect was not limited to the xenograft model because natural killer cell-dependent killing of ALL by human peripheral blood mononuclear cells (PBMCs) was also increased by CpG ODN stimulation. These results suggest that CpG ODNs have potential as therapeutic agents for the treatment of ALL.

CpG stimulation of precursor B-lineage acute lymphoblastic leukemia induces a distinct change in costimulatory molecule expression and shifts allogeneic T cells toward a Th1 response.

Immunostimulatory DNA containing unmethylated cytosine-phosphate-guanosine (CpG) induces the development of T helper 1 (Th1) immune responses. The response of B cells to CpG stimulation involves increased proliferation, cytokine production, and costimulatory molecule expression. Similar effects have been observed following CpG stimulation of a variety of malignant B cells. Pediatric precursor B acute lymphoblastic leukemia (B-ALL) cells express low levels of costimulatory molecules and are generally poor stimulators of T-cell responses. In this study, we evaluated the impact of CpG stimulation on precursor B-ALL cell lines and pediatric patient-derived samples. The ability to respond to CpG oligodeoxynucleotides was determined by the level of Toll-like receptor 9 (TLR9) expression. In contrast to both nonleukemic B-cell precursors and mature B cells, the response of precursor B-ALL cells was characterized by increased CD40 expression but only small changes in CD86 levels and no induction of CD80 expression. CpG stimulation of ALL blasts produced increased levels of interleukin-6 (IL-6), IL-8, and IL-10 but no detectable IL-12p70 and led to a skewing of allogeneic T cells, with enhanced interferon gamma (IFN-gamma) production and reduced secretion of IL-5. These results demonstrate the functional relevance of CpG stimulation of precursor B-ALL cells and provide a rational basis for study of these agents for use in treatment of this disease.

Progression of spontaneous autoimmune diabetes is associated with a switch in the killing mechanism used by autoreactive CTL.

Autoimmune (type 1) diabetes mellitus results from the destruction of insulin-producing pancreatic beta-cells by T lymphocytes. Beta-cell death that is induced by autoreactive CTL in diabetes involves both Fas/Fas ligand (FasL)- and perforin/granzyme-mediated pathways, although their relative contributions during the progression of the disease remain unknown. We demonstrate here that despite the preferential use of the Fas/FasL pathway for cytolysis of beta-cell targets, transgenic beta-cell-specific CTL were able to kill targets via the perforin pathway when triggered by a higher affinity stimulus. In addition, we show that the killing mechanism used by islet-associated CD8(+) T cells from non-obese diabetic mice changed as the mice aged and correspondingly, with the stage of diabetes. These results provide direct evidence for age-related changes in the cytotoxic pathways used by diabetogenic T cells during the progression of autoimmune diabetes.

T-cell epitopes in type 1 diabetes.

Type 1 diabetes (TID) results from T-cell-mediated destruction of pancreatic b cells in genetically predisposed individuals. Autoreactive CD4(+) T helper cells and CD8(+) cytotoxic T lymphocytes (CTLs) recognize b-cell-derived peptides in the context of major histocompatibility complex class II and I molecules, respectively, in a process that terminates in b-cell death. Many peptide epitopes derived from b-cell proteins have been described for both humans and the nonobese diabetic (NOD) mouse, but their relative importance in disease pathogenesis is unclear. The significance of identifying key b-cell epitopes is underscored by a study showing that in the NOD mouse monitoring of a single population of b-cell-specific CTLs in the peripheral blood using a high-avidity analogue of the endogenous peptide may be used to accurately predict diabetes occurrence. Future studies focused on the discovery of immunodominant b-cell epitopes and their high-avidity analogues should have considerable implications for prediction and immunotherapy of TID.

Optimization of epicutaneous immunization for the induction of CTL.

The immune system of the skin has recently been exploited for the development of non-invasive vaccine technologies. However, one of the limitations of current vaccine protocols is the inefficient priming of cytotoxic T lymphocytes (CTL). In this study, we report that the application of either an immunodominant class I MHC restricted ovalbumin peptide or whole ovalbumin protein, to tape-stripped skin together with the co-application of the bacterial enterotoxin cholera toxin (CT) induces antigen-specific CTL. Tape-stripping (TS) was found to enhance the magnitude of antibody responses to co-administered protein and to promote the generation of antigen-specific IgG(2a) responses. As well, both cholera toxin and tape-stripping enhanced epidermal dendritic cell (DC) immigration into draining lymph nodes. The adjuvant effect of co-administered cholera toxin and tape-stripping in promoting CTL priming was not dependent on IL-12. Epicutaneous immunization has previously been shown to induce robust antibody responses to administered protein antigen. We now demonstrate the induction of robust and persistent CTL responses to epicutaneously administered protein antigen. Epicutaneous immunization is cheap, simple and effective. These findings suggest the potential use of the skin for the generation of protective immune responses to both viral and tumor challenge.

Prediction of spontaneous autoimmune diabetes in NOD mice by quantification of autoreactive T cells in peripheral blood.

Autoimmune (type 1) diabetes mellitus results from the destruction of insulin-producing pancreatic beta cells by T lymphocytes. Prediction of cell-mediated autoimmune diseases by direct detection of autoreactive T cells in peripheral blood has proved elusive, in part because of their low frequency and reduced avidity for peptide MHC ligands. This article was published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.