Automation and data processing with the immucor Galileo (R) system in a university blood bank

Background: The implementation of automated techniques improves the workflow and quality of immuno-hematological results. The workflows of our university blood bank were reviewed during the implementation of an automated immunohematological testing system. Methods: Work impact of blood grouping and subgrouping, cross- matching and antibody search using the Immucor Galileo system was compared to the previous used standard manual and semi- automated methods. Results: The redesign of our workflow did not achieve a significant reduction of the specimen's working process time, the operator's time however was reduced by 23%. Corresponding results were achieved for blood grouping, Rhesus typing, antibody screen and for autocontrol when changing from two semi- automated to the Galileo system. Because of the higher sensitivity of the Immucor antibody detection system, the rate of the initial positive antibody screens rose from 4 to 6% Conclusion: The Immucor Galileo system automates routine blood bank testing with high reliability, specificity and higher sensitivity compared to our previous used standard manual and semi- automated methods

The Paired Availability Design for Historical Controls

BACKGROUND: Although a randomized trial represents the most rigorous method of evaluating a medical intervention, some interventions would be extremely difficult to evaluate using this study design. One alternative, an observational cohort study, can give biased results if it is not possible to adjust for all relevant risk factors. METHODS: A recently developed and less well-known alternative is the paired availability design for historical controls. The paired availability design requires at least 10 hospitals or medical centers in which there is a change in the availability of the medical intervention. The statistical analysis involves a weighted average of a simple "before" versus "after" comparison from each hospital or medical center that adjusts for the change in availability. RESULTS: We expanded requirements for the paired availability design to yield valid inference. (1) The hospitals or medical centers serve a stable population. (2) Other aspects of patient management remain constant over time. (3) Criteria for outcome evaluation are constant over time. (4) Patient preferences for the medical intervention are constant over time. (5) For hospitals where the intervention was available in the "before" group, a change in availability in the "after group" does not change the effect of the intervention on outcome. CONCLUSION: The paired availability design has promise for evaluating medical versus surgical interventions, in which it is difficult to recruit patients to a randomized trial

Nanofiber fabrication in a temperature and humidity controlled environment for improved fibre consistency

To fabricate nanofibers with reproducible characteristics, an important demand for many applications, the effect of controlled atmospheric conditions on resulting electrospun cellulose acetate (CA) nanofibers was evaluated for temperature ranging 17.5 - 35°C and relative humidity ranging 20% - 70%. With the potential application of nanofibers in many industries, especially membrane and filter fabrication, their reproducible production must be established to ensure commercially viability.
Cellulose acetate (CA) solution (0.2 g/ml) in a solvent mixture of acetone/DMF/ethanol (2:2:1) was electrospun into nonwoven fibre mesh with the fibre diameter ranging from 150nm to 1µm.
The resulting nanofibers were observed and analyzed by scanning electron microscopy (SEM), showing a correlation of reducing average fibre diameter with increasing atmospheric temperature. A less pronounced correlation was seen with changes in relative humidity regarding fibre diameter, though it was shown that increased humidity reduced the effect of fibre beading yielding a more consistent, and therefore better quality of fibre fabrication.
Differential scanning calorimetry (DSC) studies observed lower melt enthalpies for finer CA nanofibers in the first heating cycle confirming the results gained from SEM analysis. From the conditions that were explored in this study the temperature and humidity that gave the most suitable fibre mats for a membrane purpose were 25.0°C and 50%RH due to the highest level of fibre diameter uniformity, the lowest level of beading while maintaining a low fibre diameter for increased surface area and increased pore size homogeneity. This study has highlighted the requirement to control the atmospheric conditions during the electrospinning process in order to fabricate reproducible fibre mats

Platelet kinetics after slow versus standard transfusions: A pilot study

Background. Platelet transfusion is required in the acute phase of some thrombocytopenic disorders in order to prevent potentially dangerous hemorrhages. The purpose of this study was to assess the increase in platelet count following a slow platelet transfusion. Methods. Patients suffering from thrombocytopenia due to various underlying diseases were enrolled in the prospective pilot feasibility trial and were randomly divided into two groups. Standard platelet transfusion was administered in one group, while slow transfusion was used in the other. The platelet count was examined at 1 hour, 24 hours, and 1 week following the transfusions. Results. Although the platelet count was higher following 1 hour after transfusion via the standard method, the count tended to be higher 1 week after the transfusion in the slow transfusion group. This difference, however, only turned out to be statistically significant amongst females. Conclusion. A therapy of slow platelet transfusion might be more effective for the prevention of platelet loss. Further studies will be required to strengthen this hypothesis

Effect of splenectomy on type-1/type-2 cytokine gene expression in a patient with adult idiopathic thrombocytopenic purpura (ITP)

BACKGROUND: In view of clinical observations and laboratory results that support a central role of the spleen in idiopathic thrombocytopenic purpura (ITP) pathophysiology, we studied the effect of splenectomy on type-1 and type-2 cytokine gene expression in an adult ITP case, refractory to conservative treatment. CASE PRESENTATION: The patient was subjected to splenectomy 9 months after the diagnosis with complete response, attaining platelet counts over 150 × 10(6)/L within 10 days after the operation. Two consecutive blood samples were obtained from the patient, 3 and 7 months after the splenectomy for the purposes of this study. A control group consisted of 11 healthy adults. Peripheral blood mononuclear cells were prepared from each blood sample and cultured in vitro for 8 h with the addition of the mitogens phorbol myristate acetate and ionomycin. Total cellular RNA extracted from 10(6 )cells was submitted to semiquantitave reverse transcriptase-polymerase chain reaction (RT-PCR) for the amplification of IL-2, IFN-γ, IL-4, IL-5, and IL-10 metagraphs. The PCR products were run on ethidium-stained agarose gels, photographed and quantified by densitometry. A steep decrease of type-1 cytokine expression (IL-2, IFN-γ) and their calculated sum expressing Th1 activity was observed at 7 months post-splenectomy compared to 3 months post-splenectomy, in parallel with a rise of platelet count from 190 × 10(6)/L to 265 × 10(6)/L. The change of type-2 cytokine expression (IL-4, IL-5, IL-10) was slight and the Th2 activity (IL-4+IL-5) remained largely unchanged. The Th1/Th2 ratio, that reflects the pathogenic disease-specific T-cell immune deviation, was accordingly reduced 7 months post-splenectomy (Th1/Th2 = 1.3) compared to 3 months (Th1/Th2 = 3.5). CONCLUSIONS: The reduction of the Th1/Th2 cytokine ratio that was observed over time after splenectomy was accompanied by full clinical remission. Nevertheless, the persistence of a type-1 polarization, even after several months following spleen removal, is suggestive of a more basic abnormality of the immune function in these patients

Oxaliplatin induces drug resistance more rapidly than cisplatin in H69 small cell lung cancer cells

Cisplatin produces good responses in solid tumours including small cell lung cancer (SCLC) but this is limited by the development of resistance. Oxaliplatin is reported to show activity against some cisplatin-resistant cancers but there is little known about oxaliplatin in SCLC and there are no reports of oxaliplatin resistant SCLC cell lines. Studies of drug resistance mainly focus on the cellular resistance mechanisms rather than how the cells develop resistance. This study examines the development of cisplatin and oxaliplatin resistance in H69 human SCLC cells in response to repeated treatment with clinically relevant doses of cisplatin or oxaliplatin for either 4 days or 2h. Treatments with 200ng/ml cisplatin or 400ng/ml oxaliplatin for 4 days produced sublines (H69CIS200 and H69OX400 respectively) that showed low level (approximately 2-fold) resistance after 8 treatments. Treatments with 1000ng/ml cisplatin or 2000ng/ml oxaliplatin for 2h also produced sublines, however these were not stably resistant suggesting shorter treatment pulses of drug may be more effective. Cells survived the first five treatments without any increase in resistance, by arresting their growth for a period and then regrowing. The period of growth arrest was reduced after the sixth treatment and the H69CIS200 and H69OX400 sublines showed a reduced growth arrest in response to cisplatin and oxaliplatin treatment suggesting that "regrowth resistance" initially protected against drug treatment and this was further upregulated and became part of the resistance phenotype of these sublines. Oxaliplatin dose escalation produced more surviving sublines than cisplatin dose escalation but neither set of sublines were associated with increased resistance as determined by 5-day cytotoxicity assays, also suggesting the involvement of regrowth resistance. The resistant sublines showed no change in platinum accumulation or glutathione levels even though the H69OX400 subline was more sensitive to buthionine sulfoximine treatment. The H69CIS200 cells were cross-resistant to oxaliplatin demonstrating that oxaliplatin does not have activity against low level cisplatin resistance. Relative to the H69 cells, the H69CIS200 and H69OX400 sublines were more sensitive to paclitaxel and taxotere suggests the taxanes may be useful in the treatment of platinum resistant SCLC. These novel cellular models of cisplatin and oxaliplatin resistant SCLC will be useful in developing strategies to treat platinum-resistant SCLC

Validation of a 22-Gene Genomic Classifier in Patients With Recurrent Prostate Cancer An Ancillary Study of the NRG/RTOG 9601 Randomized Clinical Trial

IMPORTANCE: Decipher (Decipher Biosciences Inc) is a genomic classifier (GC) developed to estimate the risk of distant metastasis (DM) after radical prostatectomy (RP) in patients with prostate cancer. OBJECTIVE: To validate the GC in the context of a randomized phase 3 trial. DESIGN, SETTING, AND PARTICIPANTS: This ancillary study used RP specimens from the phase 3 placebo-controlled NRG/RTOG 9601 randomized clinical trial conducted from March 1998 to March 2003. The specimens were centrally reviewed, and RNA was extracted from the highest-grade tumor available in 2019 with a median follow-up of 13 years. Clinical-grade whole transcriptomes from samples passing quality control were assigned GC scores (scale, 0-1). A National Clinical Trials Network–approved prespecified statistical plan included the primary objective of validating the independent prognostic ability of GC for DM, with secondary end points of prostate cancer–specific mortality (PCSM) and overall survival (OS). Data were analyzed from September 2019 to December 2019. INTERVENTIONS: Salvage radiotherapy (sRT) with or without 2 years of bicalutamide. MAIN OUTCOMES AND MEASURES: The preplanned primary end point of this study was the independent association of the GC with the development of DM. RESULTS: In this ancillary study of specimens from a phase 3 randomized clinical trial, GC scores were generated from 486 of 760 randomized patients with a median follow-up of 13 years; samples from a total of 352 men (median [interquartile range] age, 64.5 (60-70) years; 314 White [89.2%] participants) passed microarray quality control and comprised the final cohort for analysis. On multivariable analysis, the GC (continuous variable, per 0.1 unit) was independently associated with DM (hazard ratio [HR], 1.17; 95% CI, 1.05-1.32; P = .006), PCSM (HR, 1.39; 95% CI, 1.20-1.63; P < .001), and OS (HR, 1.17; 95% CI, 1.06-1.29; P = .002) after adjusting for age, race/ethnicity, Gleason score, T stage, margin status, entry prostate-specific antigen, and treatment arm. Although the original planned analysis was not powered to detect a treatment effect interaction by GC score, the estimated absolute effect of bicalutamide on 12-year OS was less when comparing patients with lower vs higher GC scores (2.4% vs 8.9%), which was further demonstrated in men receiving early sRT at a prostate-specific antigen level lower than 0.7 ng/mL (−7.8% vs 4.6%). CONCLUSIONS AND RELEVANCE: This ancillary validation study of the Decipher GC in a randomized trial cohort demonstrated association of the GC with DM, PCSM, and OS independent of standard clinicopathologic variables. These results suggest that not all men with biochemically recurrent prostate cancer after surgery benefit equally from the addition of hormone therapy to sRT. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT0000287

Optimal timing for managed relocation of species faced with climate change

Managed relocation is a controversial climate-adaptation strategy to combat negative climate change impacts on biodiversity. While the scientific community debates the merits of managed relocation(1-12), species are already being moved to new areas predicted to be more suitable under climate change(13,14). To inform these moves, we construct a quantitative decision framework to evaluate the timing of relocation in the face of climate change. We find that the optimal timing depends on many factors, including the size of the population, the demographic costs of translocation and the expected carrying capacities over time in the source and destination habitats. In some settings, such as when a small population would benefit from time to grow before risking translocation losses, haste is ill advised. We also find that active adaptive management(15,16) is valuable when the effect of climate change on source habitat is uncertain, and leads to delayed movement

Kinetic Pathway of Pyrophosphorolysis by a Retrotransposon Reverse Transcriptase

DNA and RNA polymerases use a common phosphoryl transfer mechanism for base addition that requires two or three acidic amino acid residues at their active sites. We previously showed, for the reverse transcriptase (RT) encoded by the yeast retrotransposon Ty1, that one of the three conserved active site aspartates (D211) can be substituted by asparagine and still retain in vitro polymerase activity, although in vivo transposition is lost. Transposition is partially restored by second site suppressor mutations in the RNAse H domain. The novel properties of this amino acid substitution led us to express the WT and D211N mutant enzymes, and study their pre-steady state kinetic parameters. We found that the kpol was reduced by a factor of 223 in the mutant, although the Kd for nucleotide binding was unaltered. Further, the mutant enzyme had a marked preference for Mn2+ over Mg2+. To better understand the functions of this residue within the Ty1 RT active site, we have now examined the in vitro properties of WT and D211N mutant Ty1 RTs in carrying out pyrophosphorolysis, the reverse reaction to polymerization, where pyrophosphate is the substrate and dNTPs are the product. We find that pyrophosphorolysis is efficient only when the base-paired primer template region is >14 bases, and that activity increases when the primer end is blunt-ended or recessed by only a few bases. Using pre-steady state kinetic analysis, we find that the rate of pyrophosphorolysis (kpyro) in the D211N mutant is nearly 320 fold lower than the WT enzyme, and that the mutant enzyme has an ∼170 fold lower apparent Kd for pyrophosphate. These findings indicate that subtle substrate differences can strongly affect the enzyme's ability to properly position the primer-end to carry out pyrophosphorolysis. Further the kinetic data suggests that the D211 residue has a role in pyrophosphate binding and release, which could affect polymerase translocation, and help explain the D211N mutant's transposition defect