Use Of Pooled Samples Research Articles

Comparing pooled and individual samples for estimation of gastrointestinal strongyles burden and treatment efficacy in small ruminants

Monitoring endoparasite burden (FEC) and treatment efficacy (FECR) is a key element of sustainable parasite control. However, the costs of the analysis often discourage their implementation by farmers and veterinary practitioners. Pooling samples is considered to be a good alternative to reduce time and monetary costs, but limited data are available on the use of pooled samples in small ruminants, especially for goats. In this study, data collected over the years in sheep and goat farms were analyzed, and results obtained from individual and pooled analysis were compared for the purposes of FEC and FECR assessment. A total of 801 individual and 134 pooled samples (composed of 3–12 individual samples) were included. For FECR testing, 2 pools of 5 samples each were created per trial and the same animals were sampled at day 0 (D0 – treatment day) and 14 days after (D14). Samples were analyzed by McMaster technique (limit of detection 20 EPG). Results from pooled and individual FEC were not significantly different (Wilcoxon signed-rank test) and correlation (Spearman’s rank test) was high for all sub-categories, although agreement (Lin’s concordance correlation) was often classified as poor. Results were not influenced by the pool size (<6 or ≥6). Interpretation of treatment efficacy between the two methods was comparable for all sheep trials, while it differed for goats in 4 out of 10 trials. Wilcoxon signed-rank test indicated a non significant difference between pooled and individual FECR. However, correlation and agreement between FECR were considerably better for sheep compared to goats, for which they were very limited, despite the correlation between FEC at D0 and D14 was always high. According to our results, pooled FECR can be a good option but the absence of 95 %CI represents a major drawbacks in the interpretation of results. Further studies on the topic for goats are needed.

Estimation of prevalence in rare disease using pooled samples

The use of pooled samples for screening infected individuals is a known procedure to reduce costs. In an estimation problem, the aim is only to determine how many individuals are infected instead of determining who is infected (classification problem). In that setting, our goal was to compare the performance of using one or two-dimensional arrays. The best performance was established according to one of the following criteria: minimizing the number of individuals or the number of tests required to attain a certain estimate accuracy. It is observed that when we want to minimize the number of individuals used, the two-dimensional procedures have a little advantage over the one-dimensional procedures. However, when the major concern is the cost, the one-dimensional procedures clearly outperform the two-dimensional procedures.

A new protocol for the simultaneous flow cytometric analysis of cytotoxicity and immunotoxicity on zebra mussel (Dreissena polymorpha) hemocytes

Immunotoxicity analysis receives a strong interest in environmental a priori and a posteriori risk assessment procedures considering the direct involvement of the immune system in the health status of organisms, populations and thus ecosystems. The freshwater mussel Dreissena polymorpha is an invasive species widely used in ecotoxicology studies and biomonitoring surveys to evaluate the impacts of contaminants on aquatic fauna. Bivalve hemocytes are the immunocompetent cells circulating in the open circulatory system of the organism. However, there is nowadays no consensus on a protocol to evaluate the immunocompetent state of this particular cell type using flow cytometry. Wild species such as D. polymorpha present several technical barriers complicating their analyze including (i) the quality and the purity of the hemolymph sample, (ii) the controversial characterization of hemocyte subpopulations and their diversity, (iii) the quantity of biological material, and (iv) the high inter-individual variability of hemocyte responses. The present work proposes several technical and analytical improvements to control the above-mentioned issues. The inclusion of sedimentation and cell detachment steps in the pre-analytical phase of the protocol substantially ameliorate the quality of the hemolymph sample as well as the accuracy of the cytometric measurements, by selecting the analyzed cells on their adhesion ability and by increasing the concentration of the analyzed events. The development of an effective triple-labeling procedure including the cellular probe Hoechst® 33342, the membrane impermeant dye propidium iodide and yellow-green fluorescent microspheres allowed the simultaneous analysis of cytotoxicity and phagocytosis activity in hemocytes. It also significantly enhanced the accuracy of hemocyte endpoint measurements by eliminating non-target events from the analysis and allowing relevant gating strategies. Finally, the use of pooled samples of hemolymph noticeably reduced inter-sample variability while providing more plasticity in the experimental design and improving the discriminating potency between treatments. The developed protocol is suitable for ex vivo exposure of hemocyte in a chemical/environmental toxicity assessment as well as for in vivo exposure in the laboratory or in situ biomonitoring surveys with few adaptations.

Biological implications of somatic DDX41 p.R525H mutation in acute myeloid leukemia.

The DDX41 gene, encoding a DEAD-box type ATP-dependent RNA helicase, is rarely but reproducibly mutated in myeloid diseases. The acquired mutation in DDX41 is highly concentrated at c.G1574A (p.R525H) in the conserved motif VI located at the C-terminus of the helicase core domain where ATP interacts and is hydrolyzed. Therefore, it is likely that the p.R525H mutation perturbs ATPase activity in a dominant-negative manner. In this study, we screened for the DDX41 mutation of CD34-positive tumor cells based on mRNA sequencing and identified the p.R525H mutation in three cases among 23 patients. Intriguingly, these patients commonly exhibited acute myeloid leukemia (AML) with peripheral blood cytopenias and low blast counts, suggesting that the mutation inhibits the growth and differentiation of hematopoietic cells. Data from cord blood cells and leukemia cell lines suggest a role for DDX41 in preribosomal RNA processing, in which the expression of the p.R525H mutant causes a certain ribosomopathy phenotype in hematopoietic cells by suppressing MDM2-mediated RB degradation, thus triggering the inhibition of E2F activity. This study uncovered a pathogenic role of p.R525H DDX41 in the slow growth rate of tumor cells. Age-dependent epigenetic alterations or other somatic changes might collaborate with the mutation to causeAML.

Use of pooled samples to assess human exposure to parabens, benzophenone-3 and triclosan in Queensland, Australia

Parabens, benzophenone-3 and triclosan are common ingredients used as preservatives, ultraviolet radiation filters and antimicrobial agents, respectively. Human exposure occurs through consumption of processed food and use of cosmetics and consumer products. The aim of this study was to provide a preliminary characterisation of exposure to selected personal care product chemicals in the general Australian population. De-identified urine specimens stratified by age and sex were obtained from a community-based pathology laboratory and pooled (n=24 pools of 100). Concentrations of free and total (sum of free plus conjugated) species of methyl, ethyl, propyl and butyl paraben, benzophenone-3 and triclosan were quantified using isotope dilution tandem mass spectrometry; with geometric means 232, 33.5, 60.6, 4.32, 61.5 and 87.7ng/mL, respectively. Age was inversely associated with paraben concentration, and females had concentrations approximately two times higher than males. Total paraben and benzophenone-3 concentrations are significantly higher than reported worldwide, and the average triclosan concentration was more than one order of magnitude higher than in many other populations. This study provides the first data on exposure of the general Australian population to a range of common personal care product chemical ingredients, which appears to be prevalent and warrants further investigation.

7th GCC Insights: incurred samples use; fit-for-purpose validation, solution stability, electronic laboratory notebook and hyperlipidemic matrix testing.

The Global CRO Council for Bioanalysis (GCC), a global independent consortium bringing together many senior level CRO representatives, was created in 2010 in order to openly discuss and share opinions on scientific and regulatory issues specific to the bioanalytical field [1]. Since its formation, the GCC members have met on a regular basis to discuss various topics and challenges faced by bioanalytical CRO companies. Several conference reports of past GCC meetings were published to share the discussions held during these events [2–4]. In addition, the GCC also published several influential White Papers on topics of widespread interest in bioanalysis [5–9]. These White Papers provide unified GCC recommendations that are helpful to the global bioanalytical community. The 7th GCC Closed Forum for Bioanalysis took place in Long Beach, CA, USA on 8 April 2013, one day before the start of the 7th Workshop on Recent Issues in Bioanalysis (WRIB). In attendance were 46 senior-level participants from six countries, representing 37 bioanalytical CRO companies/sites. This event represented a good opportunity for bioanalytical experts from CROs to share and discuss these issues of concern for the outsourcing industry. The 7th GCC Closed Forum was chaired by Mario Rocci, who started the meeting by communicating the official admonition statement, as has been done in all previous editions [1]. Prior to initiating discussions, participants introduced themselves. As in previous GCC meetings, multiple topics of 7th GCC Insights: incurred samples use; fitfor-purpose validation, solution stability, electronic laboratory notebook and hyperlipidemic matrix testing

The maintenance of anthelmintic efficacy in sheep in a Mediterranean climate

To determine how far management practices on small ruminant dairy flocks in southern Italy have influenced the development of anthelmintic resistance (AR), faecal egg count (FEC) reduction tests were run on 54 sheep farms using the FLOTAC technique with a sensitivity of 2 eggs per gram of faeces. Tests were run on groups of sheep (12–20 animals per group) using six anthelmintics administered orally: levamisole on 8 farms, ivermectin (half and full dose) on 8 farms, moxidectin on 3 farms, monepantel on 8 farms, netobimin on 22 farms (pooled samples) and albendazole on 5 farms (pooled samples). Different formulae and statistics were used to calculate FEC reductions and confidence intervals based on the presence of a control group and/or the use of pooled samples. A very high average efficacy was obtained with all anthelmintics tested as follows: levamisole 99.3% (range across farms 98–100%), ivermectin half dose 99.5% (98.0–100%), ivermectin full dose 99.9% (99.3–100%), moxidectin 100% (99.9–100%), monepantel 99.4% (97–100%), netobimin 99.1% (92–100%) and albendazole 100%. The results suggest that the development of AR in small ruminants could be limited in countries with a Mediterranean type of climate provided that refugia of the nematode populations are maintained, anthelmintic use is restricted and movement of animals is not permitted to spread resistance.

Pooled biological specimens for human biomonitoring of environmental chemicals: Opportunities and limitations

Biomonitoring has become the "gold standard" in assessing chemical exposures, and has an important role in risk assessment. The pooling of biological specimens-combining multiple individual specimens into a single sample-can be used in biomonitoring studies to monitor levels of exposure and identify exposure trends or to identify susceptible populations in a cost-effective manner. Pooled samples provide an estimate of central tendency and may also reveal information about variation within the population. The development of a pooling strategy requires careful consideration of the type and number of samples collected, the number of pools required and the number of specimens to combine per pool in order to maximise the type and robustness of the data. Creative pooling strategies can be used to explore exposure-outcome associations, and extrapolation from other larger studies can be useful in identifying elevated exposures in specific individuals. The use of pooled specimens is advantageous as it saves significantly on analytical costs, may reduce the time and resources required for recruitment and, in certain circumstances, allows quantification of samples approaching the limit of detection. In addition, the use of pooled samples can provide population estimates while avoiding ethical difficulties that may be associated with reporting individual results.

Pooled nasopharyngeal and oropharyngeal samples for the identification of respiratory viruses in adults

A pooled sample of oropharyngeal swabs, nasopharyngeal swabs and nasopharyngeal washings, taken from each of 1,000 subjects, was compared to separate specimens from the same sampling. Multiplex real-time polymerase chain reaction (mqRT-PCR) was used to identify 12 respiratory viruses. Two hundred and forty-three (97%) of the 251 viruses identified in the separate samples were also identified in the mixed samples. The sensitivity rate was identical at 100% for all virus groups except coronaviruses. This sensitivity rate clearly justifies the use of pooled samples instead of separate samples for clinical and epidemiological purposes. The reduction in costs attained from the use of pooled samples may represent a critical advantage when considering its use in extensive clinical and epidemiological studies.

Addressing the problems of using the polymerase chain reaction technique as a stand-alone test for detecting pathogens in aquatic animals

The polymerase chain reaction (PCR) test is increasingly being used as the diagnostic tool of choice and with pathogens of aquatic animals, particularly molluscs and crustaceans, a PCR test is often the only laboratory diagnostic test available. When a diagnostic decision is required in the absence of clinical disease (e.g. health certification or a survey for disease freedom) limitations with the PCR technique may become apparent. A review of validated PCR tests demonstrates that most are imperfect assays with diagnostic sensitivities and/or specificities less than 100%. False positive and false negative results will thus occur and large numbers of samples need to be tested to achieve statistically significant results. The results from a survey for disease freedom from white spot syndrome virus in Australia are used to illustrate the problem and to assess various solutions. The use of pooled samples or the use of two independent PCR tests can substantially reduce the number of tests required and the associated costs of such a survey.

To pool or not to pool, from whether to when: applications of pooling to biospecimens subject to a limit of detection

Pooling of biological specimens has been utilised as a cost-efficient sampling strategy, but cost is not the unique limiting factor in biomarker development and evaluation. We examine the effect of different sampling strategies of biospecimens for exposure assessment that cannot be detected below a detection threshold (DT). The paper compares use of pooled samples to a randomly selected sample from a cohort in order to evaluate the efficiency of parameter estimates. The proposed approach shows that a pooling design is more efficient than a random sample strategy under certain circumstances. Moreover, because pooling minimises the amount of information lost below the DT, the use of pooled data is preferable (in a context of a parametric estimation) to using all available individual measurements, for certain values of the DT. We propose a combined design, which applies pooled and unpooled biospecimens, in order to capture the strengths of the different sampling strategies and overcome instrument limitations (i.e. DT). Several Monte Carlo simulations and an example based on actual biomarker data illustrate the results of the article.

Antibody Protein Array Analysis of the Tear Film Cytokines

Many bioactive proteins including cytokines are reported to increase in dry eye disease although the specific profile and concentration of inflammatory mediators varies considerably from study to study. In part, this variability results from inherent difficulties in quantifying low abundance proteins in a limited sample volume using relatively low sensitivity dot ELISA methods. Additional complexity comes with the use of pooled samples collected using a variety of techniques and intrinsic variation in the diurnal pattern of individual tear proteins. The current study describes a recent advance in the area of proteomics that has allowed the identification of dozens of low abundance proteins in human tear samples. Commercially available stationary phase antibody protein arrays were adapted to improve suitability for use in small volume biological fluid analysis with particular emphasis on tear film proteomics. Arrays were adapted to allow simultaneous screening for a panel of inflammatory cytokines in low volume tear samples collected from individual eyes. A preliminary study comparing tear array results in a small population of Sjögren's syndrome patients was conducted. The multiplex microplate array assays of cytokines in tear fluid present an unanticipated challenge due to the unique nature of tear fluid. The presence of factors that exhibit an affinity for plastic, capture antibodies and IgG and create a complex series of matrix effects profoundly impacting the reliability of dot ELISA, including with elevated levels of background reactivity and reduction in capacity to bind targeted protein. Preliminary results using tears collected from patients with Sjögren's syndrome reveal methodological advantages of protein array technology and support the concept that autoimmune-mediated dry eye disease has an inflammatory component. They also emphasize the inherent difficulties one can face when interpreting the results of micro-well arrays that result from blooming effects, matrix effects, image saturation and cross-talk between capture and probe antibodies that can greatly reduce signal-to-noise and limit the ability to obtain meaningful results.

The use of age-clustered pooled faecal samples for monitoring worm control in horses

A study was performed on two horse farms to evaluate the use of age-clustered pooled faecal samples for monitoring worm control in horses. In total 109 horses, 57 on farm A and 52 on farm B, were monitored at weekly intervals between 6 and 14 weeks after ivermectin treatment. This was performed through pooled faecal samples of pools of up to 10 horses of the groups ‘yearlings’ (both farms), ‘2-year-old’ (two pools in farm A), ‘3-year-old’ (farm A) and adult horses (four pools on farm A and five pools on farm B), which were compared with the mean individual faecal egg counts of the same pools. A very high correlation between the faecal egg counts in pooled samples and the mean faecal egg counts was seen and also between the faecal egg counts in pooled samples and larval counts from pooled faecal larval cultures. Faecal egg counts increased more rapidly in yearlings and 2-year-old horses than in older horses. This implied that in these groups of young animals faecal egg counts of more than 200 EPG were reached at or just after the egg reappearance period (ERP) of 8 weeks that is usually indicated for ivermectin. This probably means that, certainly under intensive conditions, repeated treatment at this ERP is warranted in these young animals, with or without monitoring through faecal examination. A different situation is seen in adult animals. Based on the mean faecal egg counts on both farms and on the results of pooled samples in farm A, using 100 EPG as threshold, no justification for treatment was seen throughout the experimental period. However, on farm B values of 100 EPG were seen at 9 and 11, 13 and 14 and 14 weeks after ivermectin treatment in pools 10, 12 and 13, respectively. This coincided with the presence of one or two horses with egg counts above 200 EPG. The conclusion is that random pooled faecal samples of 10 adult horses from a larger herd, starting at the ERP and repeating it at, for instance, 4-week intervals, could be used for decisions on worm control. However, there would be a certain risk for underestimating pasture contamination through missing high-egg excreters. An alternative use of pooled samples would be as a cheap first screening to detect which adult horses really contribute to pasture contamination with worm eggs on a farm. All horses should be sampled and subsequently animals from ‘positive’ pools can be reexamined individually.

Geometric mean estimation from pooled samples

Biomonitoring for environmental chemicals presents various challenges due to the expense of measuring some compounds and the fact that in some samples the levels of many compounds may be below the limit of detection (LOD) of the measuring instrument. Even though various statistical methods have been developed to address issues associated with data being censored because results were below the LOD, the expense of measuring many compounds in large numbers of subjects remains a challenge. One solution to these challenges is to use pooled samples. There are many problems associated with the use of pooled samples as compared with individual samples, but using pooled samples can sometimes reduce the number of analytical measurements needed. Also, because pooled samples often have larger sample volumes, using pooled samples can result in lower LODs and thereby decrease the likelihood that results will be censored. However, many data sets obtained from environmental measurements have been shown to have a log-normal distribution, so using pooled samples presents a new problem: The measured value for a pooled sample is comparable to an arithmetic average of log-normal results and thus represents a biased estimate of the central tendency of the samples making up the pool. In this paper, we present a method for correcting the bias associated with using data from pooled samples with a log-normal distribution. We use simulation experiments to demonstrate how well the bias-correction method performs. We also present estimates for levels of PCB 153 and p, p′-DDE using data from pooled samples from the 2001 to 2002 National Health and Nutrition Examination Surveys.

Metabolic Profiles for Evaluation of the Transition Period

Metabolic profiling as originally defined using the Compton Metabolic Profile procedure was insufficient for identifying herd problems. More recent research has better characterized metabolic dynamics around the time of calving and their relationship with high prevalence of metabolic and infectious disease problems. This, coupled with a series of factors including improved diagnostic tests, increasing herd size and recognition of transition cow health impact on herd performance, have revived interest in new testing protocols to assess herd periparturient disease risk. Cost and interpretation have been the limiting factors to widespread adoption of a metabolic profiling procedure. New strategies to reduce costs and yet maintain some useful diagnostic value have evolved either herd-based diagnostic or screening approaches. Use of pooled samples collected from pre-defined physiologic groups, and testing a wide array of analytes, have been advocated as a practical and economical method of screening the dairy herd for periparturient disease risk. Key diagnostic analytes identified as predictive for transition cow problems include non-esterified fatty acids (NEFA), NEFA:cholesterol ratio; B-hydroxybutyrate, albumin, calcium, sodium and magnesium. The current challenge is development of diagnostically valid interpretation criteria for pooled samples. Most importantly, it must be remembered that metabolic profiles are almost useless without being coupled with animal and facility evaluations, body condition scoring and ration evaluation. The combination, used within a team approach, can be an extremely useful diagnostic tool in nutritional evaluations of the dairy herd.

Use and Interpretation of Pooled Metabolic Profiles for Evaluating Transition Cow Health Status

Use of metabolic profiles for evaluating herd nutritional or health status has not been widely accepted in the United States. Costs associated with metabolic profiling have been a primary deterrent, as well as interpretation. In a traditional profile protocol eight to 12 individuals are sampled within a herd or animal group for evaluation. Profile results are then interpreted as a mean value or percent of individuals deviating from some defined values. Use of pooled samples was evaluated as a method to collect usable information on herd metabolic status without the high cost of individual sampling. Objectives of this study were to determine if blood metabolite concentrations from pooled serum samples were different from arithmetic mean results of individual samples, and to develop preliminary guidelines for interpretation of pooled samples.

Improvement of Barley yellow dwarf virus-PAV detection in single aphids using a fluorescent real time RT-PCR

One of the major factors determining the incidence of Barley yellow dwarf virus (BYDV) on autumn-sown cereals is the viruliferous state of immigrant winged aphids. This variable is assessed routinely using the enzyme-linked immunosorbant assay (ELISA). However, the threshold for virus detection by ELISA can lead to false negative results for aphids carrying less than 10 6 particles. Although molecular detection techniques enabling the detection of lower virus quantities in samples are available, the relatively laborious sample preparation and data analysis have restricted their use in routine applications. A gel-free real-time one-step reverse transcription polymerase chain reaction (RT-PCR) protocol is described for specific detection and quantitation of BYDV-PAV, the most widespread BYDV species in Western Europe. This new assay, based on TaqMan® technology, detects and quantifies from 10 2 to 10 8 BYDV-PAV RNA copies. This test is 10 and 10 3 times more sensitive than the standard RT-PCR and ELISA assays published previously for BYDV-PAV detection and significantly improves virus detection in single aphids. Extraction of nucleic acids from aphids using either phenol/chloroform or chelatin resin-based protocols allow the use of pooled samples or of a small part (up to 1/1600th) of a single aphid extract for efficient BYDV-PAV detection.

A Critique of Methods of Sampling and Reporting Pathogens in Populations of Fish

We used maximum likelihood methods to estimate an observed or apparent prevalence for a pathogen in a pooled sample of fish and here provide the program code for such calculations using commonly available statistical software. To illustrate the characteristics and variability of prevalence estimates from pooled samples, we explored the relationships among pathogen prevalence, sample size, and method of pooling samples. We calculated the average width of confidence intervals and the mean square error of the prevalence estimator for samples from populations with pathogen prevalence ranging from 1% to 90% using several pooling strategies for samples of 30 and 60 fish. As an illustration, we calculated the confidence interval and apparent prevalence of Myxobolus cerebralis in samples of fish from Utah screened with pooled sampling strategies. When all pools were positive, the apparent prevalence was 100%, but the bounds of the confidence interval ranged from 8% to 100%. Interpretations of data sets that are based only on the results for positive pools may be misleading, as the percentage of pools that is positive when any single pool scores negative is higher than the maximum likelihood estimate of apparent prevalence. The confidence intervals bounding estimates were generally smaller when larger numbers of groups were used and samples had few fish per pool. In populations with higher prevalence, the use of pooled samples significantly enlarges the confidence interval of estimates.

An evaluation of the use of pooled samples in studies of genetic variation.

When using molecular markers to study genetic variation, either the sampled individuals can be analysed individually or the individuals can be pooled and only the pools analysed (pooled samples). A theoretical investigation was carried out into the use of pooled samples in the detection of alleles and providing maximum likelihood estimates of allele frequency. The results show that, in many cases, pooled samples are more efficient than samples of individuals. Of the different pool sizes studied, small pools containing two or three individuals showed the smallest expected squared error of allele frequency estimates.

Differences in Organic Surface Microlayers from an Artificially Acidified and Control Lake, Elucidated by XAD-8/XAD-4 Tandem Separation and Solid State 13C NMR Spectroscopy

Solid state 13C NMR spectroscopy was used to quantify the distribution of C functional groups in surface microlayer dissolved organic matter (DOM, <0.45 μm) from an artificially acidified and a control freshwater lake in western Norway. Organic substances were extracted from pooled surface microlayers and collected with hydrophilic Teflon coated drum, using XAD-8/XAD-4 tandem separation. DOM was recovered at 80−85%. Presence of 15−20% carbohydrates in the humic and fulvic acid fractions of the acid-treated microlayer indicated greater influence of soil humic substances on microlayer DOM, compared with the control. Humic and fulvic acids and hydrophobic neutrals were enriched (ratio surface microlayer, subsurface water greater than 1) in the surface microlayers of both basins. Aromatic fractions were reduced in the microlayer from the treated basin compared to the control. The dissimilarities are primarily believed to result from hydrological and geomorphologic differences between the lake basins, secondarily from acid degradation of the aromatic DOM in the microlayers. Higher aliphatic C in the acid treated than in the control surface microlayer sup ported the degradation theory, as did the lowered aromaticity found for all fractions in the treated microlayer. Major influences were decreased (from 46 to 36%) fulvic acid fraction and lowered aromaticity of all DOM fractions in the treated surface microlayer. The combined use of XAD-resin separation and 13C NMR spectroscopy for quantification of DOM from different surface microlayers proved useful for the comparative purposes of this environmental impact study. Some caution has to be taken to the use of pooled samples, however, and the results need to be collected simultaneously with in situ measurements, such as on-line fourier transfer infra-red spectroscopy.