Time Of Blood Collection Research Articles

Diagnostic assessment of reproductive status in white-spotted bamboo sharks (Chiloscyllium plagiosum)

Relatively little is known about elasmobranch reproductive physiology compared to other species. An increased understanding of elasmobranch reproduction would improve the success of captive breeding and may aid in situ conservation efforts by reducing demand for wild-caught individuals. The reproductive status of seven adult female white-spotted bamboo sharks (Chiloscyllium plagiosum) (WSB) was monitored via coelomic ultrasonography and analysis of plasma biochemistry and steroid hormones over 6 months. Based on ultrasonic findings, females were categorized at each blood collection time point as: no follicular activity (INACTIVE), follicles but no eggs present (ACTIVE-OVARY), and eggs present within the oviduct (ACTIVE-OVIDUCT). Triglyceride concentrations were greater for those with the ACTIVE-OVARY (75.98 mg/dL; CI 61.81–90.15 mg/dL) and ACTIVE-OVIDUCT (87.0 mg/dL; CI 70.20 to 103.81 mg/dL) categories than INACTIVE (51.81 mg/dL; CI 37.07–66.54 mg/dL) category. No significant differences were observed for PCV, total solids, calcium, phosphorus, iron or progesterone. Estradiol concentrations were less for the INACTIVE (0.15 ng/ml; CI 0.08 to 0.25 ng/ml) than ACTIVE-OVARY (0.63 ng/ml; CI 0.42 to 0.88 ng/ml) and ACTIVE-OVIDUCT (0.92 ng/ml; CI 0.64–1.26 ng/ml) category. Testosterone concentrations differed among reproductive states, being greater with the INACTIVE (0.22 ng/ml; CI 0.13 to 0.37 ng/ml) and peaking in the ACTIVE-OVIDUCT (2.12 ng/ml; CI 1.25–3.60 ng/ml) state. The ultrasonic technique was performed in a standardized manner, and the anatomy was validated via opportunistic post-mortem examinations and MRI. Using the described diagnostic techniques, reproductive status in WSB can be routinely monitored, and findings have implications for improving the success of captive breeding efforts in other elasmobranch species.

Abstract 5643: TCRβ repertoire analysis from a prophylactic MUC1 cancer vaccine trial

Abstract Mucin 1 (MUC1) is a large transmembrane glycoprotein that is overexpressed and hypoglycosylated in many adenocarcinomas, including colon cancer. In the hypoglycosylated state, the peptide core of the 20aa variable number of tandem repeats (VNTR) region is exposed, allowing it to function as an altered-self ligand for antibodies and T cells. VNTR peptides have been previously used as antigens in therapeutic cancer vaccines. A clinical trial was conducted to test immunogenicity and safety of a MUC1 100mer VNTR peptide+polyICLC adjuvant vaccine in a prophylactic setting in patients with premalignant colon adenomas who were at high risk for later developing colon cancer. In this premalignant setting, where patients were expected to have fewer immunosuppressive countermeasures usually present in patients with cancer, nearly half of the individuals generated IgG antibodies with no noted adverse effects. Because the antibody response was isotype-switched, it suggested simultaneous T cell activation. To determine whether changes in T cell clonal abundance following immunization could be detected, we sequenced and analyzed TCRβ repertoires from PBMCs of 21 antibody-responders collected before and after both primary and one-year booster vaccinations. Of the patients analyzed, we identified 8 TCRβ sequences from three patients that were significantly elevated after both priming and boosting immunizations, potentially identifying T cell clones that expanded in response to the vaccine. 16 TCRβ sequences from four individuals were significantly decreased at both time points, potentially identifying T cell clones that left circulation to participate in immune responses in secondary lymphoid organs or tissues. The timing of blood collection at two weeks post immunization may have been too late to allow us to detect all of the vaccine-expanded clones before they left circulation. In summary, we found 24 TCRβ CDR3 sequences from 7/21 vaccine responders that significantly changed in frequency after both primary and booster vaccinations. TCR sequencing studies from patients' T cells expanded in vitro with MUC1 peptide will be performed to determine if they overlap with those sequenced ex vivo. Citation Format: Michelle L. Miller, Jason Lohmueller, John R. McKolanis, Robert Schoen, Olivera J. Finn. TCRβ repertoire analysis from a prophylactic MUC1 cancer vaccine trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5643.

Abstract 942: Sensitive detection of microsatellite instability (MSI) in tumors and liquid biopsies using nuclease-based enrichment

Abstract The role of MSI in colorectal cancer (CRC) is well characterized, and colon tumors are classified as MSI-High or MSI-Stable by screening specific microsatellites. MSI-H compared to MSI-S is predictive for therapy outcome in chemotherapy and immunotherapy and has been associated with distinct characteristics and favorable results including better prognosis, a higher 5-year survival, and lesser metastasis. Several other tumors also present MSI changes reflecting mismatch repair deficiency. While tumor testing is the gold standard, a convenient approach to screen for MSI before and during cancer treatment is screening circulating DNA (liquid biopsy) using a blood draw, thereby interrogating ‘systemic' MSI reflecting primary or secondary (occult) tumor status at the time of blood collection. However, presence of excess unaltered, wild-type DNA often masks alterations such as MSI. Using capillary electrophoresis or next generation sequencing for MSI detection presents challenges at low levels of MSI due to polymerase slippage (‘stutter') that generate high false positive rates at positions of homo-polymers. We present a new approach for enrichment of altered micro-satellites prior to DNA-amplification thereby facilitating their detection. We recently developed nuclease-assisted minor-allele enrichment with probe-overlap, a single-step approach that removes WT-DNA and enriches mutation-containing alleles. Here we adapted NaME-PrO for detection of homopolymer indels, for MSI detection. The method employs a double-strand-DNA-specific nuclease and overlapping oligonucleotide-probes interrogating multiple micro-satellite targets. Following DNA denaturation, the probes form double-stranded regions with their targets, thereby guiding nuclease digestion to selected sites. Microsatellite indels create ‘bulges' that inhibit digestion, thus subsequent amplification yields DNA with microsatellite alterations enhanced at multiple targets. The assay is applied at the genomic or circulating-DNA level prior to amplification, thereby avoiding polymerase-introduced ‘stutter peaks' arising from WT DNA. Inclusion of organic solvents allows homogeneous application of the method in closed tube reactions. We validated the method by evaluating 5 MSI targets simultaneously, NR27, NR21, NR24, BAT25 and BAT26 using DNA from tumor biopsies and circulating-DNA from colorectal cancer patients. The technique enriched all altered targets and detected microsatellite alterations down to 0.01% altered allele frequency, thus improving detection sensitivity by >100-fold relative to current approaches. We anticipate application of this highly- multiplex-able method either with standard 5-plex reactions in conjunction with capillary electrophoresis or with NGS-based detection of MSI on thousands of targets to enable sensitive detection in tumors and liquid biopsies. Citation Format: Ioannis Ladas, Chen Song, Fangyan Yu, Ka Wai Leong, Katerina Troullinou, Matthew Kulke, Harvey Mamon, Mike G. Makrigiorgos. Sensitive detection of microsatellite instability (MSI) in tumors and liquid biopsies using nuclease-based enrichment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 942.

Whole blood microRNAs as potential biomarkers in post-operative early breast cancer patients

BackgroundmicroRNAs (miRNAs) are considered promising cancer biomarkers, showing high reliability, sensitivity and stability. Our study aimed to identify associations between whole blood miRNA profiles, presence of circulating tumor cells (CTCs) and clinical outcome in post-operative early breast cancer patients (EBC) to assess the utility of miRNAs as prognostic markers in this setting.MethodA total of 48 post-operative patients, recruited in frame of the SUCCESS A trial, were included in this retrospective study and tested with a panel of 8 miRNAs (miR-10b, −19a, − 21, − 22, −20a, − 127, − 155, −200b). Additional 17 female healthy donors with no previous history of cancer were included in the study as negative controls. Blood samples were collected at different time points (pre-adjuvant therapy, post-adjuvant therapy, 2 years follow up), total RNA was extracted and the relative concentration of each miRNA was measured by quantitative PCR and compared in patients stratified on blood collection time or CTC detection. Furthermore, we compared miRNA profiles of patients, for each time point separately, and healthy donors. CTCs were visualized and quantified with immunocytochemistry analysis. Data were analyzed using non-parametric statistical tests.ResultsIn our experimental system, miR-19a, miR-22 and miR-127 showed the most promising results, differentiating patients at different time points and from healthy controls, while miR-20a, miR-21 and miR-200b did not show any difference among the different groups. miR-10b and miR-155 were never detectable in our experimental system. With respect to patients’ clinical characteristics, we found a significant correlation between miR-200b and lymph node status and between miR-20a and tumor type. Furthermore, miR-127 correlated with the presence of CTCs. Finally, we found a borderline significance between Progression Free Survival and miR-19a levels.ConclusionsThis pilot study suggests that profiling whole blood miRNAs could help to better stratify post-operative EBC patients without any sign of metastasis to prevent later relapse or metastatic events.

Prediagnostic Antibodies to Serum p53 and Subsequent Colorectal Cancer.

Background: The presence of circulating antibodies to the p53 tumor suppressor protein is a potential early detection colorectal cancer biomarker. However, studies of prediagnostic measures of p53 seropositivity in relation to colorectal cancer risk are limited.Methods: We conducted a nested case-control study of serum p53 autoantibodies and risk of colorectal cancer within the Cancer Prevention Study-II Nutrition Cohort. Among cohort participants who were cancer free at the time of blood collection, 392 were subsequently diagnosed with colorectal cancer over 11 years of follow-up. Two controls were matched to each case on birth date, blood draw date, race, and sex. Autoantibodies to p53 were detected in 41 of the 392 cases (10.5%) and 49 of the 774 controls (6.3%).Results: Participants who were seropositive for p53 antibodies before diagnosis were more likely to be subsequently diagnosed with colorectal cancer [RR = 1.77; 95% confidence interval (CI), 1.12-2.78]. This association was strongest within 3 years of diagnosis (RR = 2.26; 95% CI, 1.06-4.83). An association was also suggested when colorectal cancer was diagnosed 4 to <6 years after p53 measurement (RR = 1.84; 95% CI, 0.89-3.79), but not 6 or more years later (RR = 1.15; 95% CI, 0.44-2.99).Conclusions: If these results are confirmed, serum p53 antibodies may be useful on a panel of early detection markers for colorectal cancer.Impact: Individuals who were seropositive for p53 antibodies were twice as likely to develop colorectal cancer within the next 3 years compared with those who were seronegative. This marker is a good candidate for inclusion on an early detection marker panel for colorectal cancer. Cancer Epidemiol Biomarkers Prev; 27(2); 219-23. ©2017 AACR.

Changing the Time of Blood Collection to Determine Vancomycin Concentrations in Intensive Care Unit Patients

Clinical practice guidelines for initiation and therapeutic drug monitoring, but not timing, of vancomycin dosing exist at many institutions. Scheduling vancomycin trough measurements and doses around the morning blood sample collection could yield more interpretable troughs and increase patient safety. To evaluate the appropriateness of blood sample collection times for vancomycin trough measurements before and after an initiative to change the timing of blood sampling to determine vancomycin doses and trough levels in a medical intensive care unit. A retrospective cohort study was conducted of patients in a medical intensive care unit who received intravenous vancomycin at a scheduled interval. Differences in continuous and categorical data were compared between pre- and postintervention groups. The primary outcome was proportion of blood samples collected for vancomycin trough measurements within 30 minutes of the next scheduled vancomycin dose. Baseline characteristics were similar between the preintervention (n = 68) and postintervention (n = 176) groups except for the percentage of blood samples drawn for trough measurements and morning laboratory tests (6% vs 81%; P < .001). Frequency of loading doses was similar between patients in the pre- and postintervention groups, as was weight-based maintenance dosing. There was no significant difference in the percentage of blood samples collected to measure vancomycin trough levels appropriately at 30, 60, or 75 minutes from the next scheduled dose. Measuring vancomycin trough levels in morning blood samples did not affect the percentage of inappropriately collected blood samples used to measure vancomycin trough levels.

Abstract A05: Circulating tumor DNA levels correlate with response to treatment in LMS patients

Abstract Circulating tumor DNA (ctDNA) has significant potential for several clinical applications, including assessment of treatment response and monitoring of recurrent/residual disease. We performed a pilot study to explore the feasibility of ctDNA monitoring in patients with leiomyosarcoma (LMS). We profiled matching plasma and FFPE tumor specimens from 9 LMS patients. We analyzed between 2 to 6 longitudinal plasma samples (median of 5) and between 1 to 7 tumor specimens (median of 2) per patient. ctDNA analysis was performed on plasma samples collected pre-/post-surgery, throughout chemo-/radiotherapy and during follow-up. We used two separate approaches in our study: 1) targeted deep sequencing of ctDNA, tumor DNA and germline DNA to detect single nucleotide variants and indels using Cancer Personalized Profiling by deep Sequencing with integrated digital error suppression (CAPP-Seq; with a median deduplicated depth of sequencing of 2,136x); 2) copy number variant analysis in ctDNA by genome representation profiling (GRP; median coverage across the whole genome 0.23x) and in the matched tumors by SNP arrays. One patient was excluded from the analysis due to inadequate sequencing coverage in tumor specimen. For CAPP-Seq analysis, we designed a custom 184kb capture panel targeting 89 genes that are recurrently mutated in LMS. Using strict variant calling criteria (requiring that variants be present on each strand of the original DNA “duplex” molecule) our panel identified a median of one nonsynonymous coding/splicing variant per tumor. We detected the same variants in TP53, RB1 and ATRX genes in ctDNA of 6/8 patients (with a baseline sensitivity of 87.5% and overall specificity of 98.96% calculated using plasma from 24 healthy donors). These six patients presented with advanced disease at the time of the first blood collection and were progressing throughout multiple lines of therapy. Two patients who did not have any variants detectable by CAPP-Seq in plasma had localized disease at the time of the first blood collection and/or responded well to the therapy. We found that changes in ctDNA levels appear to correspond with the extent of disease and response to treatment. Specifically, ctDNA levels decreased in a subset of patients after surgery or at the time of temporary response to chemo- and/or radiotherapy. Congruently, increases in ctDNA levels correlated with progression in most of the patients. There was a high correlation between ctDNA levels detected by CAPP-Seq (quantified as mutant molecules/mL plasma) and GRP (quantified as percent of genome showing copy number aberrations) across all plasma samples (Pearson's r= 0.88, p &amp;lt; 0.0001), but in a few samples ctDNA was detected by only one of the two assays. Our results suggest that serial analysis of ctDNA is a promising approach for evaluation of treatment response in LMS patients. Validation of these findings in a prospective study on a larger group of patients will be required to determine the use of this approach in a clinical setting.

Assessment of newborn screening in the public health system of a municipality in northern Rio Grande do Sul

Introduction: Newborn screening allows the screening of diseases that are still in the asymptomatic period and whose early diagnosis and treatment are associated with reduced infant morbidity and mortality. The aim of this study was to evaluate the public National Newborn Screening Program in the municipality of Carazinho, state of Rio Grande do Sul (RS), Brazil. Methods: This was a population-based, retrospective, descriptive study. We collected and transcribed data from a database of the Carazinho municipal laboratory, which is affiliated with the referral center for newborn screening in RS. The records of all individuals undergoing newborn screening from 2005 to 2010 were reviewed, and information was collected on the program coverage, time elapsed between birth and screening (first collection), and test results. Results: The program had a coverage of 75.5%. One suspected case of phenylketonuria, three suspected cases of congenital hypothyroidism and no suspected cases of hemoglobinopathy were identified. In addition, there were 18 positive results for hemoglobin S heterozygosity, five for hemoglobin D heterozygosity, two for hemoglobin C heterozygosity, and one for a rare variant hemoglobin. When analyzing the newborn’s age at the time of blood collection, it was observed that 63.1% were within the recommended age range. Conclusion: Our findings suggest the need for optimization of public newborn screening in the evaluated municipality. The strategies to be adopted should include education of the population and especially of managers and health professionals about the importance of newborn screening. Keywords: Newborn screening; public health; mass screening

Effect of Blood Collection Tube Type and Time to Processing on the Enumeration and High-Content Characterization of Circulating Tumor Cells Using the High-Definition Single-Cell Assay.

- As circulating tumor cell (CTC) assays gain clinical relevance, it is essential to address preanalytic variability and to develop standard operating procedures for sample handling in order to successfully implement genomically informed, precision health care. - To evaluate the effects of blood collection tube (BCT) type and time-to-assay (TTA) on the enumeration and high-content characterization of CTCs by using the high-definition single-cell assay (HD-SCA). - Blood samples of patients with early- and advanced-stage breast cancer were collected into cell-free DNA (CfDNA), EDTA, acid-citrate-dextrose solution, and heparin BCTs. Time-to-assay was evaluated at 24 and 72 hours, representing the fastest possible and more routine domestic shipping intervals, respectively. - We detected the highest CTC levels and the lowest levels of negative events in CfDNA BCT at 24 hours. At 72 hours in this BCT, all CTC subpopulations were decreased with the larger effect observed in high-definition CTCs and cytokeratin-positive cells smaller than white blood cells. Overall cell retention was also optimal in CfDNA BCT at 24 hours. Whole-genome copy number variation profiles were generated from single cells isolated from all BCT types and TTAs. Cells from CfDNA BCT at 24-hour TTA exhibited the least noise. - Circulating tumor cells can be identified and characterized under a variety of collection, handling, and processing conditions, but the highest quality can be achieved with optimized conditions. We quantified performance differences of the HD-SCA for specific preanalytic variables that may be used as a guide to develop best practices for implementation into patient care and/or research biorepository processes.

CXCL10/CXCR3 axis is associated with disease activity and the development of mucocutaneous lesions in patients with Beh\xe7et\u2019s disease

The objective of this study was to investigate CXC chemokines and its receptor in patients with Behcet’s disease (BD) and their associations with disease activity. Blood samples were collected from 109 BD patients and 36 age- and sex-matched healthy controls (HCs). Twenty-two follow-up blood samples were collected in BD patients. Serum CXC chemokines (CXCL1, CXCL8, CXCL9, CXCL10, CXCL12, CXCL13 and CXCL16) and cell surface marker expression (CD3, CD4 and CXCR3) in peripheral blood mononuclear cells (PBMCs) were assayed. Clinical features including disease activity were evaluated at the time of blood collection. CXCR3 expression in skin and intestinal lesions from BD patients and HCs was assessed via immunohistochemistry. Serum CXCL10 levels were correlated with disease activity in terms of Behçet’s Disease Current Activity Form (BDCAF) (p < 0.001). In follow-up BD patients, changes in serum CXCL10 levels tended to be correlated with those of BDCAF. The percentage of CXCR3 expression in CD3-positive cells in PBMCs was inversely correlated with serum CXCL10 levels in BD patients (p = 0.022). By immunohistochemistry, the number of CXCR3-positive mononuclear cells was higher in skin and intestinal lesions of BD patients than in those of HCs. These results suggest that the CXCL10/CXCR3 axis may contribute to the pathogenesis of BD.

Influence of diurnal variation and fasting on serum iron concentrations in a community-based population

ObjectivesSerum iron is an important clinical test to help identify cases of iron deficiency or overload. Fluctuations caused by diurnal variation and diet are thought to influence test results, which may affect clinical patient management. We examined the impact of these preanalytical factors on iron concentrations in a large community-based cohort. Design and MethodsSerum iron concentration, blood collection time, fasting duration, patient age and sex were obtained for community-based clinical testing from the Laboratory Information Service at Calgary Laboratory Services for the period of January 2011 to December 2015. ResultsA total of 276,307 individual test results were obtained. Iron levels were relatively high over a long period from 8:00 to 15:00. Mean concentrations were highest at blood collection times of 11:00 for adult men and 12:00 for adult women and children, however iron levels peaked as late as 15:00 in teenagers. With regard to fasting, iron levels required approximately 5h post-prandial time to return to a baseline, except for children and teenage females where no significant variation was seen until after 11h fasting. After 10h fasting, iron concentrations in all patient groups gradually increased to higher levels compared to earlier fasting times. ConclusionsSerum iron concentrations remain reasonably stable during most daytime hours for testing purposes. In adults, blood collection after 5 to 9h fasting provides a representative estimate of a patient's iron levels. For patients who have fasted overnight, i.e. ≥12h fasting, clinicians should be aware that iron concentrations may be elevated beyond otherwise usual levels.

Seroprevalence and risk factors for Coxiella burnetii, the causative agent of Q fever in the dromedary camel (Camelus dromedarius) population in Algeria.

Query (Q) fever is a globally distributed zoonotic disease caused by Coxiella burnetii, a bacterial agent for which ruminants are the most prevalent natural reservoir. Data regarding Q fever infection in camels in Algeria are limited. Therefore, a survey to detect seroprevalence of C. burnetii antibodies was conducted among healthy camel populations in a vast area in southeastern Algeria to determine distribution of the Q fever causative organism and to identify risk factors associated with infection. Between January and March 2016, blood samples were collected from 184 camels and serum samples were subsequently analysed using a commercial Enzyme-Linked Immunosorbent Assay (ELISA) kit. At the time of blood collection, a questionnaire investigating 13 potential predisposing factors associated with C. burnetii seropositivity was completed for every dromedary camel and herd. Results were analysed by a chi-square (χ2) test and multivariate logistic regression. The seroprevalence of C. burnetii at the animal level was 71.2% (95% CI: 65.2–78.3) and 85.3% (95% CI: 72.8–97.8) at the herd level. At the animal level, differences in seroprevalence were observed because of herd size, animal age, animal sex, presence of ticks and contact with other herds. A multivariable logistic regression model identified three main risk factors associated with individual seropositivity: (1) age class > 11 years (OR = 8.81, 95% CI: 2.55–30.41), (2) herd size > 50 head (OR = 4.46, 95% CI: 1.01–19.59) and (3) infestation with ticks (OR 2.2; 95% CI: 1.1–4.5). This study of seroprevalence of C. burnetii infection in camels in Algeria revealed a high seroprevalence of Q fever in camel populations in southeastern Algeria and provided strong evidence that Q fever represents an economic, public health and veterinary concern. Appropriate measures should be taken to prevent the spread of C. burnetii and to reduce the risk of Q fever in farm animals and humans in this agro-ecologically and strategically important region of North Africa.

Toxoplasmosis, leptospirosis, and brucellosis seroepidemiology in veterinary medical students and their relation with unique health

Toxoplasmosis, leptospirosis, and brucellosis are global zoonoses, with humans as accidental participants in their transmission cycles. The can also be considered occupational diseases, because certain professionals are at greater risk of contact and infection by such zoonoses. These three diseases have different epidemiological characteristics because of the distinct environmental, social, cultural, and economic conditions where these pathogens circulate. Because of the importance of these diseases and their associations with specific occupations, we performed a seroepidemiological survey of Toxoplasma, Leptospira, and Brucella antibodies, with an analysis of the association between positive serum and certain occupational and environmental variables, in students of a veterinary medicine course (1st, 2nd, 3rd, 4th, and 5th year) in a university in the northwestern region in the state of Parana, Brazil. From May to November 2014, blood samples were collected from 157 volunteers by professionals trained in nursing and biomedicine from the same university as the veterinary students. At the time of blood collection, the students did not present any clinical signs of the three diseases of interest. To detect anti-Toxoplasma gondii, anti-Leptospira spp., and anti-Brucella antibodies, indirect immunofluorescence test (IIF), microscopic agglutination test (MAT), fast agglutination test (a screening test), and enzyme-linked immunosorbent assay (ELISA) were performed. To determine variables associated with these infections, the students were interviewed to complete an epidemiological questionnaire with environmental, behavioral, and occupational information. The associations between these variables and infections were assess by chi-square or Fischer's exact tests, with a 5% significance level (?). Of the 157 serum samples analyzed, 29.29% reacted to Toxoplasma antigens, with titers ranging from 16 to 4096 by IIF, 1.27% to Leptospira antigens, with titers ranging from 100 to 800 by MAT using Hardjo and Wolffi serovars, and 0.63% to Brucella antigens by ELISA; however, no variables were found to be associated with infection with any of these pathogens. The results of this study show that one-third of the students in the veterinary medicine course were exposed to Toxoplasma gondii, Leptospira spp., and Brucella spp. at some stage in their lives; however, it is not possible to determine whether these infections were acquired at the university, because no associations between occupational risk variables and these infections were found. An understanding of the transmission of each etiological agent and methods to prevent infection is important to maintain low prevalence levels of these zoonotic diseases during the veterinary medicine course and extra-curricular internships, when there is increased exposure to these pathogens.

A Prospective Study of Inflammatory Markers and Risk of Endometriosis.

Much evidence suggests a role for inflammation in the pathogenesis of endometriosis. Although investigators in numerous case-control studies have found elevation of inflammatory markers in patients with endometriosis, results were not consistent, and no prior prospective study is known to exist. We conducted a case-control study nested within the Nurses' Health Study II in which we examined associations between levels of plasma inflammatory markers (interleukin-1 beta, interleukin-6, soluble tumor necrosis factor α receptors 1 and 2, and high-sensitivity C-reactive protein) and the risk of laparoscopically confirmed endometriosis. From blood collections in 1996-1999 and 2007, we ascertained 350 cases patients with incident endometriosis and 694 matched controls. Women with interleukin-1 beta levels in quintiles 2-4 had a higher risk of endometriosis (for the second quintile, relative risk (RR) = 3.30, 95% confidence interval (CI): 1.06, 10.3; for the third quintile, RR = 3.36, 95% CI: 1.09, 10.4; and for the fourth quintile, RR = 4.64, 95% CI: 1.58, 13.6; P for trend = 0.62), which suggested an association beginning at 0.47 pg/mL or greater. A significant nonlinear association with levels of soluble tumor necrosis factor α receptor 2 was observed, with elevated risk of endometriosis at concentrations greater than 3,400 pg/mL. Plasma interleukin-6, soluble tumor necrosis factor α receptor 1, and high-sensitivity C-reactive protein levels were not associated with endometriosis risk. Further research in larger studies with younger age at blood collection and longer time from blood to surgical diagnosis are required to confirm these associations.

Cranial electrotherapy stimulation affects mood state but not levels of peripheral neurotrophic factors or hypothalamic- pituitary-adrenal axis regulation.

Cranial electrotherapy stimulation (CES) is reported to aid in relieving symptoms of depression and anxiety, though the mechanism underlying this effect remains unclear. Therefore, the present study aimed to evaluate changes in the hypothalamic-pituitary-adrenal (HPA) axis response and levels of neurotrophic factors, as well as changes in mood state, in patients undergoing CES therapy. Fifty healthy postmenopausal women were randomly assigned to either a Sham CES group (n = 25) or an Active CES group (n = 25). CES treatment was conducted in 20-minute sessions, three times per week for 8 weeks, using a micro current cranial electrotherapy stimulator. Blood samples were collected prior to and following the 8-week treatment period for measurement of cortisol, adrenocorticotropic hormone (ACTH), brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF) levels. Changes in mood state were also examined at the time of blood collection using the Profile of Mood States (POMS). No significant differences in cortisol, ACTH, BDNF, or NGF were observed between the two participant groups (p > 0.05) following the treatment period. However, those in the Active CES group exhibited significantly decreased Tension-Anxiety and Depression-Dejection scores on the POMS relative to pre-treatment scores (p < 0.05). Furthermore, Depression-Dejection scores following treatment were significantly lower in the Active CES group than in the Sham CES group (p < 0.05). No significant differences were observed in any other POMS scores such as Anger-Hostility, Vigor-Activity, Fatigue-Inertia, and Confusion-Bewilderment (p > 0.05). These results suggest that 8 weeks of CES treatment does not induce changes in blood levels of neurotrophic factors or HPA-axis-related hormones, though such treatment may be effective in treating symptoms of anxiety and depression.

Evaluation of liquid biopsies for molecular profiling in patients with advanced non-small cell lung cancer (NSCLC) in the relapse treatment setting.

11532 Background: Molecular profiling is limited by access to sufficient tumor tissue for comprehensive analysis and due to tumor heterogeneity, the complete range of tumor DNA abnormalities may not be represented nor accurately reflect the clinical evolution of disease. Circulating tumor DNA (ctDNA) can be used as a minimally-invasive liquid biopsy for the detection, quantification and monitoring of molecular abnormalities for personalized treatment strategies. Methods: In a prospectively designed program, to date, we have recruited 227 advanced NSCLC patients having received prior therapy, with unknown molecular profile at time of blood collection. Blood collections (10ml K2-EDTA) were performed to assess molecular profile prior to or at time of relapse. Repeat samples were performed on patients initiated on treatment and followed for up to 18months. Patient samples were analyzed with InVision (enhanced tagged-amplicon sequencing) using a 34 gene panel. Interim analysis performed with full descriptive summary statistical analyses to be presented at conference. Results: ctDNA profiling detected somatic mutations in 182pts (80.2%), predominantly located in TP53 (46%), EGFR (28%), KRAS (11%) and STK11 (7%, half of which had concurrent KRAS). Of note, clinically actionable mutations were detected: T790M (25pts, median 1.4% AF), ERBB2 (8 pts), MET (8pts) and BRAF (4pts) providing eligibility for new therapy options. 20pts including 12 EGFR/T790M+ve were evaluated for ctDNA monitoring up to 18 months (median 10m); correlation between dynamic change in mutation allele fraction and clinical response was observed, especially predictive of relapse to treatment. 10 patients demonstrated SD/PR response to osimertinib treatment with T790M detection at low allele fraction (7/10 &lt; 1% AF with 1pt at 0.08% AF). Conclusions: ctDNA can be used as a non-invasive ‘liquid biopsy’ for molecular profiling of NSCLC patients to detect clinically relevant and actionable mutations when tissue biopsy is unavailable. Liquid biopsies can be repeated as needed where tissue is not feasible, providing real-time information to support personalised treatment.

Biotinidase deficiency: Genotype-biochemical phenotype association in Brazilian patients.

IntroductionThe association between the BTD genotype and biochemical phenotype [profound biotinidase deficiency (BD), partial BD or heterozygous activity] is not always consistent. This study aimed to investigate the genotype-biochemical phenotype association in patients with low biotinidase activity.MethodsAll exons, the 5'UTR and the promoter of the BTD gene were sequenced in 72 Brazilian individuals who exhibited low biotinidase activity. For each patient, the expected biochemical phenotype based on the known genotype was compared with the observed biochemical phenotype. Additional non-genetic factors that could affect the biotinidase activity were also analysed.ResultsMost individuals were identified by neonatal screening (n = 66/72). When consecutive results for the same patient were compared, age, prematurity and neonatal jaundice appeared to affect the level of biotinidase activity. The biochemical phenotype at the time of the second blood collection changed in 11/22 patients compared to results from the first sample. Three novel variants were found: c.1337T>C (p.L446P), c.1466A>G (p.N489S) and c.962G>A (p.W321*). Some patients with the same genotype presented different biochemical phenotypes. The expected and observed biochemical phenotypes agreed in 68.5% of cases (concordant patients). The non-coding variants c.-183G>A, c.-315A>G and c.-514C>T were present in heterozygosis in 5/17 discordant patients. In addition, c.-183G>A and c.-514C>T were also present in 10/37 concordant patients.ConclusionsThe variants found in the promoter region do not appear to have a strong impact on biotinidase activity. Since there is a disparity between the BTD genotype and biochemical phenotype, and biotinidase activity may be affected by both genetic and non-genetic factors, we suggest that the diagnosis of BD should be based on more than one measurement of plasma biotinidase activity. DNA analysis can be of additional relevance to differentiate between partial BD and heterozygosity.

High-sensitive C-reactive protein and risk of incident type 2 diabetes: a case\u2013control study nested within the Singapore Chinese Health Study

BackgroundThe liver-derived C-reactive protein (CRP) is a sensitive and systemic biomarker of inflammation, and has been associated with increased risk of developing type 2 diabetes in populations other than Chinese. Therefore, we prospectively examined the relation between plasma levels of CRP and risk of type 2 diabetes (T2D) among a Chinese population.MethodsPlasma high-sensitive CRP (hs-CRP) concentrations were assayed among 571 T2D cases and 571 controls nested in the prospective cohort of the Singapore Chinese Health Study. Both cases and controls were free of physician-diagnosed diabetes, cardiovascular disease and cancer at blood collections (1999–2004). Incident physician-diagnosed T2D cases were self-reported during the follow-up visits (2006–2010), and controls were matched for age (±3 years) and date (±6 months) of blood collection and gender. Multivariable logistic regression models were used to compute the odds ratio (OR) and the corresponding 95% confidence intervals (CIs).ResultsThe mean (SD) concentrations of hs-CRP were 2.79 (2.65) and 1.86 (2.03) mg/L, respectively, in cases and controls (P < 0.001). After multivariate adjustment for T2D risk factors such as lifestyle, body mass index, plasma triglycerides and HDL cholesterol, the OR comparing the extreme quartiles of hs-CRP was 1.74 [95% CI 1.12–2.70; P for trend = 0.016]. When the analysis was limited to 279 cases who had HbA1c ≥6.5% at the time of blood collection and their controls, the OR comparing the extreme quartiles of hs-CRP was 2.43 (95% CI 1.25–4.71; P for trend = 0.003). When confined to the other 292 subjects with HbA1c <6.5% and their controls, the corresponding OR was 1.24 (95% CI 0.64–2.39; P for trend = 0.93).ConclusionsWe found that CRP was not associated with increased risk of incident diabetes in this cohort of Chinese in Singapore. Previous positive findings from prospective studies might be partly due to undiagnosed T2D among the cases during blood collection.

ABCC3 Polymorphisms and mRNA Expression Influence the Concentration of a Carboxylic Acid Metabolite in Patients on Clopidogrel and Aspirin Therapy.

Acetylsalicylic acid (ASA) and clopidogrel combined therapy has been reported to be beneficial in patients with acute coronary syndrome (ACS). Antiplatelet drug resistance, especially to clopidogrel, is a multifactorial phenomenon that affects a large number of ACS patients. The genetic contribution to this drug response is not fully elucidated. We investigated the relationship of ABC-type efflux subfamily C member 3 (ABCC3) polymorphisms and mRNA expression with plasma concentrations of clopidogrel, salicylic acid (SA) and a carboxylic acid metabolite (CAM). Clopidogrel, CAM and SA plasma concentrations were measured simultaneously by liquid chromatography-tandem mass spectrometry (LCMS/MS) from 83 ACS patients undergoing percutaneous coronary intervention. ABCC3 (rs757421, rs733392 and rs739923) and CYP2C19*2 (rs4244285) polymorphisms as well as mRNA expression were evaluated. A positive correlation was found between CAM concentrations and ABCC3 mRNA expression (r = 0.494, p < 0.0001). Patients carrying genotype AA (rs757421 variant) had higher CAM concentrations and ABCC3 mRNA expression as compared to those of GG + GA carriers (p = 0.017). A multiple linear regression analysis revealed that ABCC3 mRNA expression (p = 0.017), rs757421 AA genotype (p = 0.001), blood collection time (p = 0.018) and clopidogrel dose (p = 0.001) contributed to the concentration of CAM. No associations were observed for the CYP2C19*2 polymorphism. These results suggest that up-regulation of ABCC3 mRNA expression leads to increased plasma CAM levels through MRP3-mediated cell efflux. The ABCC3 rs757421 polymorphism may contribute to gene expression. Therefore, ABCC3 may be a potential biomarker for the response to clopidogrel.

Impact of Pre-Analytical Time on the Recovery of Pathogens from Blood Cultures: Results from a Large Retrospective Survey.

BackgroundPrompt identification of bloodstream pathogens is essential for optimal management of patients. Significant changes in analytical methods have improved the turnaround time for laboratory diagnosis. Less attention has been paid to the time elapsing from blood collection to incubation and to its potential effect on recovery of pathogens. We evaluated the performance of blood cultures collected under typical hospital conditions in relation to the length of their pre-analytical time.MethodsWe carried out a large retrospective study including 50,955 blood cultures collected, over a 30-month period, from 7,035 adult septic patients. Cultures were accepted by the laboratory only during opening time (Mon-Fri: 8am–4pm; Sat: 8am–2pm). Samples collected outside laboratory hours were stored at room temperature at clinical wards. All cultures were processed by automated culture systems. Day and time of blood collection and of culture incubation were known for all samples.ResultsA maximum pre-analytical interval of 2 hours is recommended by guidelines. When the laboratory was open, 57% of cultures were processed within 2 h. When the laboratory was closed, 4.9% of cultures were processed within 2 h (P<0.001). Samples collected when the laboratory was closed showed pre-analytical times significantly longer than those collected when laboratory was open (median time: 13 h and 1 h, respectively, P<0.001). The prevalence of positive cultures was significantly lower for samples collected when the laboratory was closed compared to open (11% vs 13%, P<0.001). The probability of a positive result decreased of 16% when the laboratory was closed (OR:0.84; 95%CI:0.80–0.89, P<0.001). Further, each hour elapsed from blood collection to incubation resulted associated with a decrease of 0.3% (OR:0.997; 95%CI:0.994–0.999, P<0.001) in the probability of a positive result.DiscussionDelayed insertions of cultures into automated systems was associated with lower detection rates, with potentially important consequences for patients. In each hospital setting the logistic factors able to shorten pre-analytical time should be carefully investigated and specifically targeted.