The metabolome of any given biological system contains a diverse range of low molecular weight molecules (metabolites), whose abundances can be affected by the timing and method of sample collection, ...storage, and handling. Thus, it is necessary to consider the requirements for preanalytical processes and biobanking in metabolomics research. Poor practice can create bias and have deleterious effects on the robustness and reproducibility of acquired data.
This review presents both current practice and latest evidence on preanalytical processes and biobanking of samples intended for metabolomics measurement of common biofluids and tissues. It highlights areas requiring more validation and research and provides some evidence-based guidelines on best practices.
Although many researchers and biobanking personnel are familiar with the necessity of standardizing sample collection procedures at the axiomatic level (e.g., fasting status, time of day, "time to freezer," sample volume), other less obvious factors can also negatively affect the validity of a study, such as vial size, material and batch, centrifuge speeds, storage temperature, time and conditions, and even environmental changes in the collection room. Any biobank or research study should establish and follow a well-defined and validated protocol for the collection of samples for metabolomics research. This protocol should be fully documented in any resulting study and should involve all stakeholders in its design. The use of samples that have been collected using standardized and validated protocols is a prerequisite to enable robust biological interpretation unhindered by unnecessary preanalytical factors that may complicate data analysis and interpretation.
Abstract
Context
Adrenal venous sampling (AVS) is the key test for subtyping primary aldosteronism (PA), but its interpretation varies widely across referral centers and this can adversely affect the ...management of PA patients.
Objectives
To investigate in a real-life study the rate of bilateral success and identification of unilateral aldosteronism and their impact on blood pressure outcomes in PA subtyped by AVS.
Design and settings
In a retrospective analysis of the largest international registry of individual AVS data (AVIS-2 study), we investigated how different cut-off values of the selectivity index (SI) and lateralization index (LI) affected rate of bilateral success, identification of unilateral aldosteronism, and blood pressure outcomes.
Results
AVIS-2 recruited 1625 individual AVS studies performed between 2000 and 2015 in 19 tertiary referral centers. Under unstimulated conditions, the rate of biochemically confirmed bilateral AVS success progressively decreased with increasing SI cut-offs; furthermore, with currently used LI cut-offs, the rate of identified unilateral PA leading to adrenalectomy was as low as <25%. A within-patient pairwise comparison of 402 AVS performed both under unstimulated and cosyntropin-stimulated conditions showed that cosyntropin increased the confirmed rate of bilateral selectivity for SI cut-offs ≥ 2.0, but reduced lateralization rates (P < 0.001). Post-adrenalectomy outcomes were not improved by use of cosyntropin or more restrictive diagnostic criteria.
Conclusion
Commonly used SI and LI cut-offs are associated with disappointingly low rates of biochemically defined AVS success and identified unilateral PA. Evidence-based protocols entailing less restrictive interpretative cut-offs might optimize the clinical use of this costly and invasive test. (J Clin Endocrinol Metab XX: 0-0, 2020)
Self-collection of cervico-vaginal samples for human papillomavirus (HPV) testing has the potential to make cervical cancer screening more accessible to underscreened women. We evaluated the ...acceptability and ease of use of home-based HPV self-collection within a diverse population of low-income, infrequently screened women.
Participants were low-income women from North Carolina who had not received Pap testing in 4 or more years. Eligible women received a self-collection kit containing instructions and a brush for home-based sample collection. A total of 227 women returned a self-collected sample by mail and completed a questionnaire to assess their experiences with HPV self-collection. We described acceptability measures and used logistic regression to identify predictors of overall positive thoughts about the self-collection experience.
Nearly all women were willing to perform HPV self-collection again (98%) and were comfortable receiving the self-collection kit in the mail (99%). Overall, 81% of participants reported positive thoughts about home-based self-collection. Women with at least some college education and those who were divorced, separated or widowed were more likely to report overall positive thoughts. Aspects of self-collection that participants most commonly reported liking included convenience (53%), ease of use (32%) and privacy (23%). The most frequently reported difficulties included uncertainty that the self-collection was done correctly (16%) and difficulty inserting the self-collection brush (16%).
Home-based self-collection for HPV was a highly acceptable screening method among low-income, underscreened women and holds the promise to increase access to cervical cancer screening in this high-risk population.
Laboratory testing to support the care of patients with highly infectious diseases may pose a risk for laboratory workers. However, data on the risk of virus transmission during routine laboratory ...testing conducted using standard personal protective equipment (PPE) are sparse. Our objective was to measure laboratory contamination during routine analysis of patient specimens. Remnant specimens were spiked with the nonpathogenic bacteriophage MS2 at 1.0 × 10
PFU/ml, and contamination was assessed using reverse transcriptase PCR (RT-PCR) for MS2. Specimen containers were exteriorly coated with a fluorescent powder to enable the visualization of gross contamination using UV light. Testing was performed by two experienced laboratory technologists using standard laboratory PPE and sample-to-answer instrumentation. Fluorescence was noted on the gloves, bare hands, and laboratory coat cuffs of the laboratory technologist in 36/36 (100%), 13/36 (36%), and 4/36 (11%) tests performed, respectively. Fluorescence was observed in the biosafety cabinet (BSC) in 8/36 (22%) tests, on test cartridges/devices in 14/32 (44%) tests, and on testing accessory items in 29/32 (91%) tests. Fluorescence was not observed on or in laboratory instrumentation or adjacent surfaces. In contrast to fluorescence detection, MS2 detection was infrequent (3/286 instances 1%) and occurred during test setup for the FilmArray instrument and on FilmArray accessory equipment. The information from this study may provide opportunities for the improvement of clinical laboratory safety practices so as to reduce the risk of pathogen transmission to laboratory workers.
The preservation of high-quality biospecimens and associated data for research purposes is being performed in variety of academic, government, and industrial settings. Often these are multimillion ...dollar operations, yet despite these sizable investments, the economics of biobanking initiatives is not well understood. Fundamental business principles must be applied to the development and operation of such resources to ensure their long-term sustainability and maximize their impact. The true costs of developing and maintaining operations, which may have a variety of funding sources, must be better understood. Among the issues that must be considered when building a biobank economic model are: understanding the market need for the particular type of biobank under consideration and understanding and efficiently managing the biobank’s “value chain,” which includes costs for case collection, tissue processing, storage management, sample distribution, and infrastructure and administration. By using these value chain factors, a Total Life Cycle Cost of Ownership (TLCO) model may be developed to estimate all costs arising from owning, operating, and maintaining a large centralized biobank. The TLCO approach allows for a better delineation of a biobank’s variable and fixed costs, data that will be needed to implement any cost recovery program. This article represents an overview of the efforts made recently by the National Cancer Institute’s Office of Biorepositories and Biospecimen Research as part of its effort to develop an appropriate cost model and cost recovery program for the cancer HUman Biobank (caHUB) initiative. All of these economic factors are discussed in terms of maximizing caHUB’s potential for long-term sustainability but have broad applicability to the wide range of biobanking initiatives that currently exist.
Fecal analyses are becoming increasingly important for equine establishments as a means of parasite surveillance and detection of anthelmintic resistance. Although several studies have evaluated ...various egg counting techniques, little is known about the quantitative effects of pre-analytic factors such as collection and storage of fecal samples. This study evaluated the effects of storage temperature, storage time and airtight versus open-air storage on fecal egg counts. The experimental protocols were replicated in two study locations: Copenhagen, Denmark and Athens, Georgia, USA. In both locations, the experiment was repeated three times, and five repeated egg counts were performed at each time point of analysis. In experiment A, feces were collected rectally and stored airtight at freezer (−10 to −18
°C), refrigerator (4
°C), room (18–24
°C), or incubator (37–38
°C) temperatures. Egg counts were performed after 0, 6, 12, 24, 48, and 120
h of storage. In experiment B, feces were collected rectally and stored airtight or in the open air in the horse barn for up to 24
h. Egg counts were performed after 0, 3, 6, 12, and 24
h of storage. In experiment A at both locations, samples kept in the refrigerator showed no decline in egg counts, whereas storage in the freezer and incubator led to significantly declining egg numbers during the study. In contrast, storage at room temperature yielded marked differences between the two study locations: egg counts remained stable in the U.S. study, whereas the Danish study revealed a significant decline after 24
h. In experiment B, the Danish study showed no differences between airtight and open-air storage and no changes over time, while the U.S. study found a significant decline for open-air storage after 12
h. This difference was attributed to the different barn temperatures in the two studies. To our knowledge, this is the first study to evaluate the pre-analytic factors affecting egg counts in horses using an experimental protocol replicated in two contrasting geographic and climatic locations. Our results demonstrate that refrigeration is the best method for storage of fecal samples intended for egg count analysis, but that accurate results can be derived from fecal samples collected from the ground within 12
h of passage.
Both polyester and foam nasal swabs were collected from convalescent COVID-19 patients at a single visit and stored in viral transport media (VTM), saline or dry. Sensitivity of each swab material ...and media combination were estimated, three by three tables were constructed to measure polyester and foam concordance, and cycle threshold (Ct) values were compared. 126 visits had polyester and foam swabs stored in viral transport media (VTM), 51 had swabs stored in saline, and 63 had a foam swab in VTM and a polyester swab stored in a dry tube. Polyester and foam swabs had an estimated sensitivity of 87.3% and 94.5% respectively in VTM, 87.5% and 93.8% respectively in saline, and 75.0% and 90.6% respectively for dry polyester and foam VTM. Polyester and foam Ct values were correlated, but polyester showed decreased performance for cases with a viral load near the detection threshold and higher Ct values on average.
Whole genome sequencing (WGS) can investigate the entire Mycobacterium tuberculosis (Mtb) genome but currently requires large amounts of mycobacterial DNA, necessitating culture. Culture-free Mtb WGS ...could revolutionize the clinical use of WGS but is hampered by the high viscosity, low mycobacterial load, and high contamination with bacterial and human DNA in sputum samples. To improve the sputum liquefaction and decontamination step prior to DNA extraction, we assessed the efficiency of Myco-TB, MycoPrep, and Sputolysin with/without TiKa-Kic in liquefying and decontaminating sputum and aimed to evaluate the effect of these approaches on mycobacterial viability, and Mtb DNA quality and quantity. Experiments using spiked sputum samples showed that Myco-TB and BD MycoPrep with standard (15 min) or increased (30 min) incubation time, but not reduced (7,5 min) incubation time performed well in liquefying and decontaminating sputum. No difference in DNA quality or quantity, contamination, or the amount of human DNA present was observed. In comparison, Sputolysin with/without TiKa-Kic was less effective for liquefaction and decontamination of sputum. PCR amplification of the human GAPDH gene after sputum treatment, showed the presence of human DNA in all samples, regardless of sputum treatment. Focused efforts are needed to deplete contaminating DNA for culture-free Mtb WGS.
•Myco-TB and BD MycoPrep have similar liquefaction and decontamination efficiency.•Sputolysin ± TiKa-Kic is not effective for sputum liquefaction and decontamination.•DNase treatment of sediments did not completely deplete human DNA.
Laboratory diagnostics are a core component of any pneumonia etiology study. Recent advances in diagnostic technology have introduced newer methods that have greatly improved the ability to identify ...respiratory pathogens. However, determining the microbial etiology of pneumonia remains a challenge, especially in children. This is largely because of the inconsistent use of assays between studies, difficulties in specimen collection, and problems in interpreting the presence of pathogens as being causally related to the pneumonia event. The laboratory testing strategy for the Pneumonia Etiology Research for Child Health (PERCH) study aims to incorporate a broad range of diagnostic testing that will be standardized across the 7 participating sites. We describe the current status of laboratory diagnostics for pneumonia and the PERCH approach for specimen testing. Pneumonia diagnostics are evolving, and it is also a priority of PERCH to collect and archive specimens for future testing by promising diagnostic methods that are currently under development.
Summary Linking psychosocial measures to the cortisol awakening response (CAR) demands accurate saliva sampling times. Monitoring adherence to the saliva sampling protocol requires electronic ...monitoring of both awakening and sampling times since self-reported times are inaccurate. Delays greater than 15 min between awakening and commencement of saliva sampling reduce CAR magnitude. Less delay has been judged tolerable but remains unexplored for different magnitude measures, and for timing of the CAR peak. Study 1: Fifty healthy females (21 ± 4 years) were instructed to collect saliva on four days at 0, 15, 30 and 45 min post-awakening (samples 1–4). Both self-reported awakening and sampling times were electronically monitored using actigraphy and track caps. Self-reported awakening was later than actigraph estimated awakening (median difference of 4 min). Estimates of CAR magnitude were significantly greater on non-adherent days (delay of 5–15 min) compared to adherent days (delay < 5 min). On non-adherent compared to adherent days cortisol on average peaked earlier, at sample 3 rather than at sample 4. Study 2: Accurately timed cortisol values were obtained in an intensive investigation of 10 participants who collected saliva on 2 days every 5 min for 30 min post-awakening. Cortisol did not significantly increase until 10 min post-awakening, suggesting a time lag may be typical between awakening and observation of a cortisol increase. We conclude that moderate delays between awakening and collection of saliva samples previously considered tolerable result in erroneous estimation of CAR magnitude and timing of the peak. These results are attributed to an approximate 10 min time lag between awakening and the start of the cortisol rise. The absence of this latent period in calculations leads to overestimation of the CAR magnitude on moderately non-adherent sampling days. These findings, if more universally generalizable, will further theoretical understanding of the physiology of the CAR, but are methodologically challenging for researchers since self-reported awakening times are not accurate enough to override the concerns raised. However accurate electronic measurement of adherence to protocol would enable sampling delays to be taken into account in computing CAR estimates.
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