Impact of a post-donation hemoglobin testing strategy on efficiency and safety of whole blood donation in England: A modeling study.

BACKGROUND: Deferrals due to low hemoglobin are time-consuming and costly for blood donors and donation services. Furthermore, accepting donations from those with low hemoglobin could represent a significant safety issue. One approach to reduce them is to use hemoglobin concentration alongside donor characteristics to inform personalized inter-donation intervals. STUDY DESIGN AND METHODS: We used data from 17,308 donors to inform a discrete event simulation model comparing personalized inter-donation intervals using "post-donation" testing (i.e., estimating current hemoglobin from that measured by a hematology analyzer at last donation) versus the current approach in England (i.e., pre-donation testing with fixed intervals of 12-weeks for men and 16-weeks for women). We reported the impact on total donations, low hemoglobin deferrals, inappropriate bleeds, and blood service costs. Personalized inter-donation intervals were defined using mixed-effects modeling to estimate hemoglobin trajectories and probability of crossing hemoglobin donation thresholds. RESULTS: The model had generally good internal validation, with predicted events similar to those observed. Over 1 year, a personalized strategy requiring ≥90% probability of being over the hemoglobin threshold, minimized adverse events (low hemoglobin deferrals and inappropriate bleeds) in both sexes and costs in women. Donations per adverse event improved from 3.4 (95% uncertainty interval 2.8, 3.7) under the current strategy to 14.8 (11.6, 19.2) in women, and from 7.1 (6.1, 8.5) to 26.9 (20.8, 42.6) in men. In comparison, a strategy incorporating early returns for those with high certainty of being over the threshold maximized total donations in both men and women, but was less favorable in terms of adverse events, with 8.4 donations per adverse event in women (7.0, 10,1) and 14.8 (12.1, 21.0) in men. DISCUSSION: Personalized inter-donation intervals using post-donation testing combined with modeling of hemoglobin trajectories can help reduce deferrals, inappropriate bleeds, and costs.

Comparison of four methods to measure haemoglobin concentrations in whole blood donors (COMPARE): A diagnostic accuracy study.

OBJECTIVE: To compare four haemoglobin measurement methods in whole blood donors. BACKGROUND: To safeguard donors, blood services measure haemoglobin concentration in advance of each donation. NHS Blood and Transplant's (NHSBT) customary method have been capillary gravimetry (copper sulphate), followed by venous spectrophotometry (HemoCue) for donors failing gravimetry. However, NHSBT's customary method results in 10% of donors being inappropriately bled (ie, with haemoglobin values below the regulatory threshold). METHODS: We compared the following four methods in 21 840 blood donors (aged ≥18 years) recruited from 10 NHSBT centres in England, with the Sysmex XN-2000 haematology analyser, the reference standard: (1) NHSBT's customary method; (2) "post donation" approach, that is, estimating current haemoglobin concentration from that measured by a haematology analyser at a donor's most recent prior donation; (3) "portable haemoglobinometry" (using capillary HemoCue); (4) non-invasive spectrometry (using MBR Haemospect or Orsense NMB200). We assessed sensitivity; specificity; proportion who would have been inappropriately bled, or rejected from donation ("deferred") incorrectly; and test preference. RESULTS: Compared with the reference standard, the methods ranged in test sensitivity from 17.0% (MBR Haemospect) to 79.0% (portable haemoglobinometry) in men, and from 19.0% (MBR Haemospect) to 82.8% (portable haemoglobinometry) in women. For specificity, the methods ranged from 87.2% (MBR Haemospect) to 99.9% (NHSBT's customary method) in men, and from 74.1% (Orsense NMB200) to 99.8% (NHSBT's customary method) in women. The proportion of donors who would have been inappropriately bled ranged from 2.2% in men for portable haemoglobinometry to 18.9% in women for MBR Haemospect. The proportion of donors who would have been deferred incorrectly with haemoglobin concentration above the minimum threshold ranged from 0.1% in men for NHSBT's customary method to 20.3% in women for OrSense. Most donors preferred non-invasive spectrometry. CONCLUSION: In the largest study reporting head-to-head comparisons of four methods to measure haemoglobin prior to blood donation, our results support replacement of NHSBT's customary method with portable haemoglobinometry.

Size-Dependent Sensitivity of Aquatic Insects to Metals.

Laboratory assessments of aqueous metal toxicity generally demonstrate aquatic insects tolerate relatively high concentrations of metals in aqueous exposures; however, mesocosm experiments and field biomonitoring often indicate effects at relatively low metal concentrations. One hypothesis proposed to reconcile this discrepancy is an increased sensitivity of smaller size classes of organisms. We exposed field colonized benthic communities to aqueous metals in a series of mesocosm experiments. In addition, a novel single-species test system was used to expose first instar, mid-instar, and late instar mayflies (Ephemeroptera, Baetis tricaudatus) to Zn. These experimental approaches tested the hypothesis that small invertebrate size classes are more sensitive than large, mature size classes. Mesocosm results demonstrated strong size-dependent responses of aquatic insects to metals. Smaller organisms generally displayed greater mortality than large, mature individuals, and models were improved when size was included as a predictor of mortality. Size-dependent responses of Baetis spp. occurred in mesocosm experiments and in our single-species test system. The median lethal concentration (LC50) for early instar B. tricaudatus was less than 6% of the previously reported LC50 for late instars. Together, these results suggest that aquatic insect body size is an important predictor of susceptibility to aqueous metals. Toxicity models that account for insect phenology by integrating the natural size progression of organisms have the potential to improve accuracy in predicting effects of metals in the field.

Mesoderm migration in Drosophila is a multi-step process requiring FGF signaling and integrin activity.

Migration is a complex, dynamic process that has largely been studied using qualitative or static approaches. As technology has improved, we can now take quantitative approaches towards understanding cell migration using in vivo imaging and tracking analyses. In this manner, we have established a four-step model of mesoderm migration during Drosophila gastrulation: (I) mesodermal tube formation, (II) collapse of the mesoderm, (III) dorsal migration and spreading and (IV) monolayer formation. Our data provide evidence that these steps are temporally distinct and that each might require different chemical inputs. To support this, we analyzed the role of fibroblast growth factor (FGF) signaling, in particular the function of two Drosophila FGF ligands, Pyramus and Thisbe, during mesoderm migration. We determined that FGF signaling through both ligands controls movements in the radial direction. Thisbe is required for the initial collapse of the mesoderm onto the ectoderm, whereas both Pyramus and Thisbe are required for monolayer formation. In addition, we uncovered that the GTPase Rap1 regulates radial movement of cells and localization of the beta-integrin subunit, Myospheroid, which is also required for monolayer formation. Our analyses suggest that distinct signals influence particular movements, as we found that FGF signaling is involved in controlling collapse and monolayer formation but not dorsal movement, whereas integrins are required to support monolayer formation only and not earlier movements. Our work demonstrates that complex cell migration is not necessarily a fluid process, but suggests instead that different types of movements are directed by distinct inputs in a stepwise manner.

Evaluation of interventions to prevent vasovagal reactions among whole blood donors: rationale and design of a large cluster randomised trial.

BACKGROUND: Vasovagal reactions (VVRs) are the most common acute complications of blood donation. Responsible for substantial morbidity, they also reduce the likelihood of repeated donations and are disruptive and costly for blood services. Although blood establishments worldwide have adopted different strategies to prevent VVRs (including water loading and applied muscle tension [AMT]), robust evidence is limited. The Strategies to Improve Donor Experiences (STRIDES) trial aims to reliably assess the impact of four different interventions to prevent VVRs among blood donors. METHODS: STRIDES is a cluster-randomised cross-over/stepped-wedge factorial trial of four interventions to reduce VVRs involving about 1.4 million whole blood donors enrolled from all 73 blood donation sites (mobile teams and donor centres) of National Health Service Blood and Transplant (NHSBT) in England. Each site ("cluster") has been randomly allocated to receive one or more interventions during a 36-month period, using principles of cross-over, stepped-wedge and factorial trial design to assign the sequence of interventions. Each of the four interventions is compared to NHSBT's current practices: (i) 500-ml isotonic drink before donation (vs current 500-ml plain water); (ii) 3-min rest on donation chair after donation (vs current 2 min); (iii) new modified AMT (vs current practice of AMT); and (iv) psychosocial intervention using preparatory materials (vs current practice of nothing). The primary outcome is the number of in-session VVRs with loss of consciousness (i.e. episodes involving loss of consciousness of any duration, with or without additional complications). Secondary outcomes include all in-session VVRs (i.e. with and without loss of consciousness), all delayed VVRs (i.e. those occurring after leaving the venue) and any in-session non-VVR adverse events or reactions. DISCUSSION: The STRIDES trial should yield novel information about interventions, singly and in combination, for the prevention of VVRs, with the aim of generating policy-shaping evidence to help inform blood services to improve donor health, donor experience, and service efficiency. TRIAL REGISTRATION: ISRCTN: 10412338. Registration date: October 24, 2019.

DNA methylation of hypertension-related genes and effect of riboflavin supplementation in adults stratified by genotype for the MTHFR C677T polymorphism.

BACKGROUND: The interaction between genetic, epigenetic and environmental factors plays an important role in the aetiology of hypertension. GWAS and observational studies link the C677T polymorphism in methylenetetrahydrofolate reductase (MTHFR) with hypertension, while riboflavin, the MTHFR cofactor, has been shown to reduce blood pressure and global DNA methylation in homozygous (TT genotype) individuals. It is currently unclear whether riboflavin modulates DNA methylation of other hypertension-related genes. OBJECTIVES: To compare DNA methylation of hypertension-related genes in adults stratified by MTHFR genotype and effect of riboflavin intervention in adults with the variant MTHFR 677TT genotype. METHOD: Pyrosequencing was carried out for hypertension-related genes (ACE, AGTR1, GCK, GNA12, IGF2, MMP9 and NOS3) in blood samples from participants in previous trials (CC, n = 40; TT, n = 40). The effect of intervention with riboflavin (1.6 mg/d for16 weeks) or placebo on DNA methylation was investigated in adults with the variant MTHFR 677TT genotype (n = 80). RESULTS: Individuals with the MTHFR 677TT v CC genotype had significantly higher average DNA methylation at NOS3 (+1.66%, P = 0.044). In response to riboflavin supplementation in TT individuals, there was an increase in average DNA methylation at IGF2 (+1.09%, P = 0.019) and a decrease at ACE (-0.44%, P = 0.021) in females only. Specific CpG sites were hypomethylated in GNA12 and hypermethylated in AGTR1. CONCLUSION: This study provides the first RCT evidence that riboflavin alters DNA methylation of hypertension-related genes in adults with the MTHFR 677TT genotype, providing some insight into mechanisms linking hypertension with the genotype-specific response of BP to riboflavin.

Application of the Theory of Planned Behavior to Predict Nutrition Students' Intention to Work with Older Adults.

As Registered Dietitian Nutritionists play an important role in providing nutrition care plans for successful aging, it is important to identify factors influencing nutrition students' intention to work with older adults. A cross-sectional study was conducted to identify significant predictors of nutrition students' intention to work with older adults by utilizing the Theory of Planned Behavior (TPB). A total of 183 students, majoring in Nutrition at a large Southern University, completed a questionnaire examining validated TPB constructs. Structural Equation Modeling with maximum likelihood was used to analyze the data. The structural model provided a good fit to the data (x2 = 44.95, df = 38, p < 0.01; RMSEA = 0.03, CFI = 0.99; TLI = 0.99. Attitude (γ = 0.49, p < 0.001) and subjective norm (γ = 0.39, p < 0.001) emerged as significant predictors of nutrition students' intention to work with older adults. Findings highlight the importance of providing dietetics curricula that provide opportunities for students to develop positive attitudes toward working with older adults. Dietetic educators should present themselves as positive role models in working with older adults, as the educators influence students' intention to work with this population.

Impact of the MTHFR C677T polymorphism on one-carbon metabolites: Evidence from a randomised trial of riboflavin supplementation.

Homozygosity for the C677T polymorphism in MTHFR (TT genotype) is associated with a 24-87% increased risk of hypertension. Blood pressure (BP) lowering was previously reported in adults with the TT genotype, in response to supplementation with the MTHFR cofactor, riboflavin. Whether the BP phenotype associated with the polymorphism is related to perturbed one-carbon metabolism is unknown. This study investigated one-carbon metabolites and their responsiveness to riboflavin in adults with the TT genotype. Plasma samples from adults (n 115) screened for the MTHFR genotype, who previously participated in RCTs to lower BP, were analysed for methionine, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), betaine, choline and cystathionine by liquid chromatography tandem mass spectrometry (LC-MS/MS). The one-carbon metabolite response to riboflavin (1.6 mg/d; n 24) or placebo (n 23) for 16 weeks in adults with the TT genotype was also investigated. Plasma SAM (74.7 ± 21.0 vs 85.2 ± 22.6 nmol/L, P = 0.013) and SAM:SAH ratio (1.66 ± 0.55 vs 1.85 ± 0.51, P = 0.043) were lower and plasma homocysteine was higher (P = 0.043) in TT, compared to CC individuals. In response to riboflavin, SAM (P = 0.008) and cystathionine (P = 0.045) concentrations increased, with no responses in other one-carbon metabolites observed. These findings confirm perturbed one-carbon metabolism in individuals with the MTHFR 677TT genotype, and for the first time demonstrate that SAM, and cystathionine, increase in response to riboflavin supplementation in this genotype group. The genotype-specific, one-carbon metabolite responses to riboflavin intervention observed could offer some insight into the role of this gene-nutrient interaction in blood pressure.

Riboflavin supplementation alters global and gene-specific DNA methylation in adults with the MTHFR 677 TT genotype.

DNA methylation is important in regulating gene expression and genomic stability while aberrant DNA methylation is associated with disease. Riboflavin (FAD) is a cofactor for methylenetetrahydrofolate reductase (MTHFR), a critical enzyme in folate recycling, which generates methyl groups for homocysteine remethylation to methionine, the pre-cursor to the universal methyl donor S-adenosylmethionine (SAM). A polymorphism (C677T) in MTHFR results in decreased MTHFR activity and increased homocysteine concentration. Previous studies demonstrated that riboflavin modulates this phenotype in homozygous adults (MTHFR 677 TT genotype), however, DNA methylation was not considered. This study examined DNA methylation, globally and at key MTHFR regulatory sites, in adults stratified by MTHFR genotype and the effect of riboflavin supplementation on DNA methylation in individuals with the 677 TT genotype. Samples were accessed from participants, screened for the MTHFR C677T polymorphism, who participated in observational (n = 80) and targeted riboflavin (1.6 mg/day) RCTs (n = 80). DNA methylation at LINE-1 and key regulatory regions of the MTHFR locus were analysed by pyrosequencing in peripheral blood leukocytes. LINE-1 (+1.6%; p = 0.011) and MTHFR south shelf (+4.7%, p < 0.001) were significantly hypermethylated in individuals with the MTHFR 677 TT compared to CC genotype. Riboflavin supplementation resulted in decreased global methylation, albeit only significant at one CpG. A significant reduction in DNA methylation at the MTHFR north shore (-1.2%, p < 0.001) was also observed in TT adults following intervention with riboflavin. This provides the first RCT evidence that DNA methylation may be modulated by riboflavin in adults with the MTHFR 677 TT genotype.

Novel Approaches to Investigate One-Carbon Metabolism and Related B-Vitamins in Blood Pressure.

Hypertension, a major risk factor for heart disease and stroke, is the world's leading cause of preventable, premature death. A common polymorphism (677C→T) in the gene encoding the folate metabolizing enzyme methylenetetrahydrofolate reductase (MTHFR) is associated with increased blood pressure, and there is accumulating evidence demonstrating that this phenotype can be modulated, specifically in individuals with the MTHFR 677TT genotype, by the B-vitamin riboflavin, an essential co-factor for MTHFR. The underlying mechanism that links this polymorphism, and the related gene-nutrient interaction, with hypertension is currently unknown. Previous research has shown that 5-methyltetrahydrofolate, the product of the reaction catalysed by MTHFR, appears to be a positive allosteric modulator of endothelial nitric oxide synthase (eNOS) and may thus increase the production of nitric oxide, a potent vasodilator. Blood pressure follows a circadian pattern, peaking shortly after wakening and falling during the night, a phenomenon known as 'dipping'. Any deviation from this pattern, which can only be identified using ambulatory blood pressure monitoring (ABPM), has been associated with increased cardiovascular disease (CVD) risk. This review will consider the evidence linking this polymorphism and novel gene-nutrient interaction with hypertension and the potential mechanisms that might be involved. The role of ABPM in B-vitamin research and in nutrition research generally will also be reviewed.

A Quantitative System for Studying Metastasis Using Transparent Zebrafish.

Metastasis is the defining feature of advanced malignancy, yet remains challenging to study in laboratory environments. Here, we describe a high-throughput zebrafish system for comprehensive, in vivo assessment of metastatic biology. First, we generated several stable cell lines from melanomas of transgenic mitfa-BRAF(V600E);p53(-/-) fish. We then transplanted the melanoma cells into the transparent casper strain to enable highly quantitative measurement of the metastatic process at single-cell resolution. Using computational image analysis of the resulting metastases, we generated a metastasis score, µ, that can be applied to quantitative comparison of metastatic capacity between experimental conditions. Furthermore, image analysis also provided estimates of the frequency of metastasis-initiating cells (∼1/120,000 cells). Finally, we determined that the degree of pigmentation is a key feature defining cells with metastatic capability. The small size and rapid generation of progeny combined with superior imaging tools make zebrafish ideal for unbiased high-throughput investigations of cell-intrinsic or microenvironmental modifiers of metastasis. The approaches described here are readily applicable to other tumor types and thus serve to complement studies also employing murine and human cell culture systems.

FGF ligands in Drosophila have distinct activities required to support cell migration and differentiation.

Fibroblast growth factor (FGF) signaling controls a vast array of biological processes including cell differentiation and migration, wound healing and malignancy. In vertebrates, FGF signaling is complex, with over 100 predicted FGF ligand-receptor combinations. Drosophila melanogaster presents a simpler model system in which to study FGF signaling, with only three ligands and two FGF receptors (FGFRs) identified. Here we analyze the specificity of FGFR [Heartless (Htl) and Breathless (Btl)] activation by each of the FGF ligands [Pyramus (Pyr), Thisbe (Ths) and Branchless (Bnl)] in Drosophila. We confirm that both Pyr and Ths can activate Htl, and that only Bnl can activate Btl. To examine the role of each ligand in supporting activation of the Htl FGFR, we utilize genetic approaches that focus on the earliest stages of embryonic development. When pyr and ths are equivalently expressed using the Gal4 system, these ligands support qualitatively different FGFR signaling responses. Both Pyr and Ths function in a non-autonomous fashion to support mesoderm spreading during gastrulation, but Pyr exhibits a longer functional range. pyr and ths single mutants exhibit defects in mesoderm spreading during gastrulation, yet only pyr mutants exhibit severe defects in dorsal mesoderm specification. We demonstrate that the Drosophila FGFs have different activities and that cell migration and differentiation have different ligand requirements. Furthermore, these FGF ligands are not regulated solely by differential expression, but the sequences of these linked genes have evolved to serve different functions. We contend that inherent properties of FGF ligands make them suitable to support specific FGF-dependent processes, and that FGF ligands are not always interchangeable.

Quantitative imaging of collective cell migration during Drosophila gastrulation: multiphoton microscopy and computational analysis.

This protocol describes imaging and computational tools to collect and analyze live imaging data of embryonic cell migration. Our five-step protocol requires a few weeks to move through embryo preparation and four-dimensional (4D) live imaging using multi-photon microscopy, to 3D cell tracking using image processing, registration of tracking data and their quantitative analysis using computational tools. It uses commercially available equipment and requires expertise in microscopy and programming that is appropriate for a biology laboratory. Custom-made scripts are provided, as well as sample datasets to permit readers without experimental data to carry out the analysis. The protocol has offered new insights into the genetic control of cell migration during Drosophila gastrulation. With simple modifications, this systematic analysis could be applied to any developing system to define cell positions in accordance with the body plan, to decompose complex 3D movements and to quantify the collective nature of cell migration.

Dynamic analyses of Drosophila gastrulation provide insights into collective cell migration.

The concerted movement of cells from different germ layers contributes to morphogenesis during early embryonic development. Using an optimized imaging approach and quantitative methods, we analyzed the trajectories of hundreds of ectodermal cells and internalized mesodermal cells within Drosophila embryos over 2 hours during gastrulation. We found a high level of cellular organization, with mesoderm cell movements correlating with some but not all ectoderm movements. During migration, the mesoderm population underwent two ordered waves of cell division and synchronous cell intercalation, and cells at the leading edge stably maintained position. Fibroblast growth factor (FGF) signaling guides mesodermal cell migration; however, we found some directed dorsal migration in an FGF receptor mutant, which suggests that additional signals are involved. Thus, decomposing complex cellular movements can provide detailed insights into collective cell migration.