Extension of platelet shelf life with an improved bacterial testing algorithm.

BACKGROUND: At Canadian Blood Services, platelet concentrate (PC) shelf life was extended to 7 days with a large-volume, delayed-sampling bacterial screening algorithm. We present the development study and postimplementation results. STUDY DESIGN AND METHODS: In the development study, PCs inoculated with five bacteria (various concentrations) were incubated for 7 days with daily sampling for BacT/ALERT cultures and bacterial quantification. After implementation, from August 2017 to December 2019, a total of 223 156 pools and 39 725 apheresis units and 5310 outdated PCs were screened. Since March 2018, cocomponents associated to false-positive results have been released to inventory. RESULTS: In the development study, Klebsiella pneumoniae, Serratia marcescens, and Staphylococcus aureus were detected at concentrations of at least 0.01 colony-forming units (CFUs)/mL at 24 hours postinoculation. However, Staphylococcus epidermidis was detected at concentrations of less than 0.16 CFUs/mL only more than 48 hours postinoculation. After implementation, 776 (0.35%) and 303 (0.77%) initial-positive results and 201 (0.09%) and 16 (0.04%) confirmed-positive results were obtained for pools and apheresis units, respectively, predominantly with Cutibacterium acnes. Other organisms included staphylococci, streptococci, Klebsiella oxytoca and Pseudomonas aeruginosa. One nonfatal reaction involving a 7-day pool contaminated with S. epidermidis occurred. Approximately, 1-in-1000 false-negative screening results were obtained during testing of outdated PCs. Approximately 1000 cocomponents associated with false-positive results were released into inventory. Combined PC outdating at Canadian Blood Services and hospitals was reduced from 18.9% to 13.1%. CONCLUSION: Screening of 7-day PCs increased bacterial detection mainly of anaerobes and reduced outdating. The incidence of septic transfusion events has decreased approximately threefold. A longer surveillance period is needed to evaluate the value of anaerobic cultures and residual safety risk.

Non-linear Entropy Analysis in EEG to Predict Treatment Response to Repetitive Transcranial Magnetic Stimulation in Depression.

Background: Biomarkers that predict clinical outcomes in depression are essential for increasing the precision of treatments and clinical outcomes. The electroencephalogram (EEG) is a non-invasive neurophysiological test that has promise as a biomarker sensitive to treatment effects. The aim of our study was to investigate a novel non-linear index of resting state EEG activity as a predictor of clinical outcome, and compare its predictive capacity to traditional frequency-based indices. Methods: EEG was recorded from 62 patients with treatment resistant depression (TRD) and 25 healthy comparison (HC) subjects. TRD patients were treated with excitatory repetitive transcranial magnetic stimulation (rTMS) to the dorsolateral prefrontal cortex (DLPFC) for 4 to 6 weeks. EEG signals were first decomposed using the empirical mode decomposition (EMD) method into band-limited intrinsic mode functions (IMFs). Subsequently, Permutation Entropy (PE) was computed from the obtained second IMF to yield an index named PEIMF2. Receiver Operator Characteristic (ROC) curve analysis and ANOVA test were used to evaluate the efficiency of this index (PEIMF2) and were compared to frequency-band based methods. Results: Responders (RP) to rTMS exhibited an increase in the PEIMF2 index compared to non-responders (NR) at F3, FCz and FC3 sites (p < 0.01). The area under the curve (AUC) for ROC analysis was 0.8 for PEIMF2 index for the FC3 electrode. The PEIMF2 index was superior to ordinary frequency band measures. Conclusion: Our data show that the PEIMF2 index, yields superior outcome prediction performance compared to traditional frequency band indices. Our findings warrant further investigation of EEG-based biomarkers in depression; specifically entropy indices applied in band-limited EEG components. Registration in ClinicalTrials.Gov; identifiers NCT02800226 and NCT01887782.

Laser capture microdissection of embryonic cells and preparation of RNA for microarray assays.

In order to compare the global gene expression profiles of different embryonic cell types, it is first necessary to isolate the specific cells of interest. The purpose of this chapter is to provide a step-by-step protocol to perform laser capture microdissection (LCM) on embryo samples and obtain sufficient amounts of high-quality RNA for microarray hybridizations. Using the LCM/microarray strategy on mouse embryo samples has some challenges, because the cells of interest are available in limited quantities. The first step in the protocol is to obtain embryonic tissue, and immediately cryoprotect and freeze it in a cryomold containing Optimal Cutting Temperature freezing media (Sakura Finetek), using a dry ice-isopentane bath. The tissue is then cryosectioned, and the microscope slides are processed to fix, stain, and dehydrate the cells. LCM is employed to isolate specific cell types from the slides, identified under the microscope by virtue of their morphology. Detailed protocols are provided for using the currently available ArcturusXT LCM instrument and CapSure(®) LCM Caps, to which the selected cells adhere upon laser capture. To maintain RNA integrity, upon removing a slide from the final processing step, or attaching the first cells on the LCM cap, LCM is completed within 20 min. The cells are then immediately recovered from the LCM cap using a denaturing solution that stabilizes RNA integrity. RNA is prepared using standard methods, modified for working with small samples. To ensure the validity of the microarray data, the quality of the RNA is assessed using the Agilent bioanalyzer. Only RNA that is of sufficient integrity and quantity is used to perform microarray assays. This chapter provides guidance regarding troubleshooting and optimization to obtain high-quality RNA from cells of limited availability, obtained from embryo samples by LCM.

Kruppel-like factor 1 (KLF1), KLF2, and Myc control a regulatory network essential for embryonic erythropoiesis.

The Krüppel-like factor 1 (KLF1) and KLF2 positively regulate embryonic ß-globin expression and have additional overlapping roles in embryonic (primitive) erythropoiesis. KLF1(-/-) KLF2(-/-) double knockout mice are anemic at embryonic day 10.5 (E10.5) and die by E11.5, in contrast to single knockouts. To investigate the combined roles of KLF1 and KLF2 in primitive erythropoiesis, expression profiling of E9.5 erythroid cells was performed. A limited number of genes had a significantly decreasing trend of expression in wild-type, KLF1(-/-), and KLF1(-/-) KLF2(-/-) mice. Among these, the gene for Myc (c-Myc) emerged as a central node in the most significant gene network. The expression of the Myc gene is synergistically regulated by KLF1 and KLF2, and both factors bind the Myc promoters. To characterize the role of Myc in primitive erythropoiesis, ablation was performed specifically in mouse embryonic proerythroblast cells. After E9.5, these embryos exhibit an arrest in the normal expansion of circulating red cells and develop anemia, analogous to KLF1(-/-) KLF2(-/-) embryos. In the absence of Myc, circulating erythroid cells do not show the normal increase in α- and ß-like globin gene expression but, interestingly, have accelerated erythroid cell maturation between E9.5 and E11.5. This study reveals a novel regulatory network by which KLF1 and KLF2 regulate Myc to control the primitive erythropoietic program.

Comparative resin kinetics using in situ fluorescence measurements.

The aminolysis of a novel activated ester resin was utilized for kinetic study via continuous in situ fluorescence measurements. A variety of resin compositions (polystyrene, JandaJel, ArgoPore, TentaGel, NovaGel, and PEGA) and solvents (dimethylformamide, acetonitrile, tetrahydrofuran, 1,2-dichlorethane, and toluene) were tested to compare their effects on the reaction rate. A linear relationship between the reaction rate and (solvent polarity x swelling of resin) was elucidated for the aminolysis reaction.