Enhanced 3-epi-25-hydroxyvitamin D3 signal leads to overestimation of its concentration and amplifies interference in 25-hydroxyvitamin D LC-MS/MS assays

Background 3-epi-25-hydroxyvitamin D3 (3-epi-25OHD3) interferes in most liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays for 25-hydroxyvitamin D (25OHD). The clinical significance of this is unclear, with concentrations from undetectable to 230 nmol/L reported. Many studies have quantified 3-epi-25OHD3 based on 25OHD3 calibrators or other indirect methods, and we speculated that this contributes to the observed variability in reported 3-epi-25OHD3 concentrations. Methods We compared continuous MS/MS infusions of 3-epi-25OHD3 and 25OHD3 solutions, spiked both analytes into the same serum matrix and analysed patient samples to assess the effect of three different quantitation methods on 3-epi-25OHD3 concentration. Experiments were performed on an LC-MS/MS system using a phenyl column which does not resolve 3-epi-25OHD3, and a modified method utilizing a Zorbax SB-CN column that chromatographically resolves 3-epi-25OHD3 from 25OHD3. Results A greater 3-epi-25OHD3 signal, compared with 25OHD3, was observed during equimolar post-column continuous infusion of analyte solutions, and following analysis of a serum pool spiked with both analytes. 3-epi-25OHD3 signal enhancement was dependent on mobile phase composition. Compared with 3-epi-25OHD3 calibrators, indirect quantitation methods resulted in up to 10 times as many samples having 3-epi-25OHD3 concentrations ≥ 10 nmol/L, and an approximately fourfold increase in the maximum observed 3-epi-25OHD3 concentration to 95 nmol/L. Conclusions Enhanced 3-epi-25OHD3 signal leads to overestimation of its concentrations in the indirect quantitation methods used in many previous studies. The enhanced signal may contribute to greater interference in some 25OHD LC-MS/MS assays than others. We highlight that equimolar responses cannot be assumed in LC-MS/MS systems, even if two molecules are structurally similar

Defining end user requirements for a field-based molecular detection system for wildlife forensic investigations

The increasing use of non-laboratory-based DNA and protein detection methods promise to provide rapid investigative intelligence and support sample prioritisation. Primarily developed for human forensic or medical applications, current systems may also show utility in the field of wildlife forensic science. However, it is currently unknown whether the requirements of the wildlife forensic community can be met by current non-laboratory based tools. Given the diverse array of stakeholders and sample types commonly encountered, it is necessary to first identify the needs of the community and then try and map their needs to current instrumentation. By using a market research style questionnaire, this study identified key requirements for a non-laboratory-based system following feedback from the wildlife forensic community. Data showed that there is strong support for field-based detection methods while highlighting concerns including contamination risks and reduced quality assurance associated with non-laboratory testing. Key species and applications were identified alongside hurdles to implementation and adoption. Broadly, the requirements align with many of the developmental drivers that have led to the rise of in-field portable detection instrumentation, specifically rapid detection within one hour, ease-of-use, and ≥95% accuracy. Several existing platforms exist that met some of the identified requirements but not all. With further collaboration between industry partners and the wildlife forensic community it is possible that new field-based systems can be developed and applied routinely

Identification of a serum biomarker panel for the differential diagnosis of cholangiocarcinoma and primary sclerosing cholagnitis

The non-invasive differentiation of malignant and benign biliary disease is a clinical challenge. Carbohydrate antigen 19-9 (CA19-9), leucine-rich α2-glycoprotein (LRG1), interleukin 6 (IL6), pyruvate kinase M2 (PKM2), cytokeratin 19 fragment (CYFRA21.1) and mucin 5AC (MUC5AC) have reported utility for differentiating cholangiocarcinoma (CCA) from benign biliary disease. Herein, serum levels of these markers were tested in 66 cases of CCA and 62 cases of primary sclerosing cholangitis (PSC) and compared with markers of liver function and inflammation. Markers panels were assessed for their ability to discriminate malignant and benign disease. Several of the markers were also assessed in pre-diagnosis biliary tract cancer (BTC) samples with performances evaluated at different times prior to diagnosis. We show that LRG1 and IL6 were unable to accurately distinguish CCA from PSC, whereas CA19-9, PKM2, CYFRA21.1 and MUC5AC were significantly elevated in malignancy. Area under the receiver operating characteristic curves for these individual markers ranged from 0.73–0.84, with the best single marker (PKM2) providing 61% sensitivity at 90% specificity. A panel combining PKM2, CYFRA21.1 and MUC5AC gave 76% sensitivity at 90% specificity, which increased to 82% sensitivity by adding gamma-glutamyltransferase (GGT). In the pre-diagnosis setting, LRG1, IL6 and PKM2 were poor predictors of BTC, whilst CA19-9 and C-reactive protein were elevated up to 2 years before diagnosis. In conclusion, LRG1, IL6 and PKM2 were not useful for early detection of BTC, whilst a model combining PKM2, CYFRA21.1, MUC5AC and GGT was beneficial in differentiating malignant from benign biliary disease, warranting validation in a prospective trial

Growth and characterization of semiconductor nanoparticles in porous sol-gel films

Published versio

Convergence of multiple markers and analysis methods defines the genetic distinctiveness of cryptic pitvipers

Using multiple markers and multiple analytical approaches is critical for establishing species boundaries reliably, especially so in the case of cryptic species. Despite development of new and powerful analytical methods, most studies continue to adopt a few, with the choice often being subjective. One such example is routine analysis of Amplified Fragment Length Polymorphism (AFLP) data using population genetic models despite disparity between method assumptions and data properties. The application of newly developed methods for analyzing this dominant marker may not be entirely clear in the context of species delimitation. In this study, we use AFLPs and mtDNA to investigate cryptic speciation in the Trimeresurus macrops complex that belongs to a taxonomically difficult lineage of Asian pitvipers. We analyze AFLPs using population genetic, phylogenetic, multivariate statistical, and Bayes Factor Delimitation methods. A gene tree from three mtDNA markers provided additional evidence. Our results show that the inferences about species boundaries that can be derived from population genetic analysis of AFLPs have certain limitations. In contrast, four multivariate statistical analyses produced clear clusters that are consistent with each other, as well as with Bayes Factor Delimitation results, and with mtDNA and total evidence phylogenies. Furthermore, our results concur with allopatric distributions and patterns of variation in individual morphological characters previously identified in the three proposed species: T. macrops sensu stricto, T. cardamomensis, and T. rubeus. Our study provides evidence for reproductive isolation and genetic distinctiveness that define these taxa as full species. In addition, we re-emphasize the importance of examining congruence of results from multiple methods of AFLP analysis for inferring species diversity

Change-point of multiple biomarkers in women with ovarian cancer

To date several algorithms for longitudinal analysis of ovarian cancer biomarkers have been proposed in the literature. An issue of specific interest is to determine whether the baseline level of a biomarker changes significantly at some time instant (change-point) prior to the clinical diagnosis of cancer. Such change-points in the serum biomarker Cancer Antigen 125 (CA125) time series data have been used in ovarian cancer screening, resulting in earlier detection with a sensitivity of 85% in the most recent trial, the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS, number ISRCTN22488978; NCT00058032). Here we propose to apply a hierarchical Bayesian change-point model to jointly study the features of time series from multiple biomarkers. For this model we have analytically derived the conditional probability distribution of every unknown parameter, thus enabling the design of efficient Markov Chain Monte Carlo methods for their estimation. We have applied these methods to the estimation of change-points in time series data of multiple biomarkers, including CA125 and others, using data from a nested case-control study of women diagnosed with ovarian cancer in UKCTOCS. In this way we assess whether any of these additional biomarkers can play a role in change-point detection and, therefore, aid in the diagnosis of the disease in patients for whom the CA125 time series does not display a change-point. We have also investigated whether the change-points for different biomarkers occur at similar times for the same patient. The main conclusion of our study is that the combined analysis of a group of specific biomarkers may possibly improve the detection of change-points in time series data (compared to the analysis of CA125 alone) which, in turn, are relevant for the early diagnosis of ovarian cancer

Sex hormone measurements using mass spectrometry and sensitive extraction radioimmunoassay and risk of estrogen receptor negative and positive breast cancer: Case control study in UK Collaborative Cancer Trial of Ovarian Cancer Screening (UKCTOCS)

INTRODUCTION: Associations of endogenous sex hormone levels and all as well as estrogen-receptor (ER)-positive breast cancers are well described. However, studies investigating their association with ER-negative tumours are limited and none use accurate assays such as mass spectrometry. METHODS: Within the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS), a nested case-control study was undertaken of postmenopausal-women who developed ER-negative (n=92) or ER-positive (n=205) breast cancer after sample donation and 297 (1:1) age-matched controls. Androgens (testosterone and androstenedione) were measured using mass spectrometry and estradiol by extraction radioimmunoassay (RIA). Bioavailable estradiol and testosterone were calculated using the total hormone level and the sex hormone-binding globulin concentration. Subjects were classified according to the quartile range among controls. Logistic regression was used to estimate odds-ratio (OR) and 95% confidence-intervals (CI) of the associations between two factors and breast cancer risk. A separate analysis was done by stratifying the women based on whether they provided their samples less than or more than 2years before diagnosis. RESULTS: Estradiol and free estradiol were significantly higher prior to diagnosis of ER-negative breast cancer compared with controls while androgens and SHBG did not show any difference. Estradiol, free estradiol, free testosterone and SHBG were significantly higher before ER-positive breast cancer diagnosis compared with controls. Women had a twofold increased ER-negative breast cancer risk if estradiol and free estradiol were in the top quartile but not androgens (testosterone and androstenedione) or SHBG. These associations remained significant only when samples closer (median 1.1y before) to diagnosis were analyzed rather than farther from diagnosis (median 2.9y before). Women had a 2.34 (95% CI: 1.21-4.61, p=0.001), 2.21 (95% CI: 1.14-4.38, p=0.001), 2 (95% CI: 1.05-3.89, p=0.005) fold increased ER-positive breast cancer risk if estradiol, free estradiol and free testosterone respectively were in the top quartile. These associations remained significant regardless of whether the samples were collected less than or more than 2years prior to diagnosis. CONCLUSION: In postmenopausal women increased estrogens but not androgens are associated with ER-negative breast cancer. Previously reported associations of estradiol and free testosterone with ER-positive breast cancer are confirmed. The use of mass spectrometry and sensitive RIA add validity to these findings

Cancer associated auto-antibodies to MUC1 and MUC4 - A blinded case control study of colorectal cancer in UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS)

Recent reports suggest that autoantibodies directed to aberrantly glycosylated mucins, in particular MUC1 and MUC4, are found in patients with colorectal cancer. There is, however, limited information on the autoantibody levels prior to clinical diagnosis, and their utility in cancer screening in the general population. In this study, we have generated O-glycosylated synthetic MUC1 and MUC4 peptides in vitro, to mimic cancer associated glycoforms, and displayed these on microarrays. The assay's performance was tested through an initial screening of serum samples taken from patients at the time of colorectal cancer diagnosis and healthy controls. Subsequently the selected biomarkers were evaluated in a blinded nested case control study, using stored serum samples from among the 50,640 women randomised to the multimodal arm of the UKCTOCS, where women gave annual blood samples for several years. Cases were 97 postmenopausal women who developed colorectal cancer following recruitment, and were age-matched to 97 women without any history of cancer. MUC1-STn and MUC1-Core3 IgG autoantibodies identified cases with 8.2% and 13.4% sensitivity, respectively, at 95% specificity. IgA to MUC4-glycoforms were unable to discriminate between cases and controls in the UKCTOCS sera. Additional analysis was undertaken by combining the data of MUC1-STn and MUC1-Core3 with previously generated data on autoantibodies to p53 peptides, which increased the sensitivity to 32.0% at 95% specificity in the UKCTOCS set. These findings suggest that a combination of antibody signatures may have a role as part of a biomarker panel for the early detection of colorectal cancer

Effect of remote ischaemic conditioning on contrast-induced nephropathy in patients undergoing elective coronary angiography (ERICCIN): rationale and study design of a randomised single-centre, double-blind placebo-controlled trial.

Contrast-induced nephropathy (CIN), an acute kidney injury resulting from the administration of intravascular iodinated contrast media, is an important cause of morbidity/mortality following coronary angiographic procedures in high-risk patients. Despite preventative measures intended to mitigate the risk of CIN, there remains a need for an effective intervention. Remote ischaemic conditioning (RIC), where non-injurious ischaemia is applied to an arm prior to the administration of contrast, has shown promise in attenuating CIN but its effectiveness in preserving long-term renal function is unknown, which will be studied as part of the effect of remote ischaemic conditioning against contrast-induced nephropathy (ERICCIN) trial. ( http://Controlled-trials.com Identifier: ISRCTN49645414.) METHODS: The ERICCIN trial is a single-centre, randomised double-blinded placebo-controlled trial which plans to recruit 362 patients who are at risk of CIN, defined by pre-existent renal impairment (estimated glomerular filtration rate 25 % of eGFR, or rise of creatinine of >44 μmol/l) at 48 h. A key secondary endpoint will be whether RIC impacts upon persistent renal impairment over the 3-month follow-up period. Additional secondary endpoints include the measurement of serum neutrophil gelatinase-associated lipocalin and urinary albumin at 6, 48 h and 3 months following administration of contrast