Use of samples from biobanks for commercial purposes – JournalClub no.053

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Exploring how biobanks communicate the possibility of commercial access and its associated benefits and risks in participant documents

Samuel G, Hardcastle F, Broekstra R, Lucassen A. Exploring how biobanks communicate the possibility of commercial access and its associated benefits and risks in participant documents. BMC Med Ethics. 2022;23:95. doi:10.1186/s12910-022-00829-1

Bio-banks and biomedical research repositories rely on volunteer participants who provide their samples and associated data. These banks aim to advance medical knowledge and improve health outcomes, but there are concerns about international sample and data sharing and relationships between public/private and non-profit/profit organizations.

How Bio-banks are Advancing Medical Knowledge and Improving Health Outcomes

While the purpose of bio-banks is to support health research, participants must be informed of the potential benefits and risks associated with their contribution. The samples collected are often used to study diseases such as cancer, diabetes, heart disease, and stroke. Some bio-banks offer participants the option to receive additional or incidental findings, which are carefully communicated and provided by a clinical practitioner.

Commercial Interaction with Bio-banks - The Question of Ethics

One issue that arises in the use of bio-banks is the question of whether they should interact with the commercial sector. While some bio-banks permit commercial organizations to access samples and data, stringent data governance policies ensure that only organizations that meet specific access requirements can access the data. However, the absence of information about commercial interactions can lead to mistrust among potential participants.

Addressing Concerns about Public/Private Interactions in Bio-banking

To address this issue, bio-banks should provide more information about the nature of public/private interactions, including the associated benefits and risks. Websites can be used to enlighten participants about these interactions, but they should not be the only means of addressing concerns. Population conversation should be centered on lay people, who should be involved in decisions about access to a bio-bank’s resources

Nuanced Approach to Risk and Benefit Communication in Bio-banking for Maintaining Sample Integrity

It is also important to recognize the blurred lines between public and private entities and activities, which can lead to polarized discourse. A nuanced approach is needed to address the important issues of risk and benefit communication in bio-banking. Sample storage conditions and preanalytics play a crucial role in maintaining the integrity of the samples and the associated data. By communicating these factors effectively, bio-banks can help participants make informed decisions about their contribution and support the advancement of medical knowledge for the public good.

Sample storage and preparation of whole milk samples for determination of microbiota – #JournalClub no.054

by JournalClubNicht kategorisiert

Improved assessments of bulk milk micro-biota composition via sample preparation and DNA extraction methods

Xue Z, Marco ML. Improved assessments of bulk milk microbiota composition via sample preparation and DNA extraction methods. PLOS ONE. 2022;17(9):e0267992. doi:10.1371/journal.pone.0267992

DNA sequencing is the most common method for studying microbes in humans, animals, food and the environment. The workflow for sample preparation and DNA sequencing consists of numerous steps that are prone to bias and contamination problems. Numerous bacterial species are present in the diverse microbiota of high-quality whole milk. Within the study by Xue et al. presented here, the following bacterial strains were used:

  • Bacillus subtilis
  • Clostridium tyrobutyricum
  • Corynebacterium bovis
  • Enterococcus faecalis
  • Escherichia coli
  • Lactococcus lactis
  • Pseudomonas fluorexcens
  • Staphylococcus aureus
  • Staphylococcus agalactiae

Bakterielle Cell-Mock-Community (BCMC)

Nine strains in BCMC1 were grown to near stationary phase and counted directly under the microscope. The BCMC2 pools just produced were either used immediately for DNA extraction or stored at -80°C. To obtain the bacteria, BCMC1 and BCMC2 were inoculated with ultra-high temperature pasteurised milk and centrifuged at 13,000 for five minutes at 4°C before DNA extraction. They were stored for seven days at -20°C in PBS or PBS containing 25% v/v glycerol.

Methods for cell lysis and DNA purification

It is possible that Escherichia coli and Pseudomonas are more sensitive to DNA shearing and lysis than Gram-positive bacteria. B5 (tapping at 4 m/sec for 10 seconds) and V3 (vortexing at 1800 rpm for 30 seconds) were two of the milder methods tried to break the cells. Three MagMAX DNA purification kits Total, Core and Ultra2 were compared and the Total kit gave the most accurate results.

Impact of the BCMC preparation method on the estimation of community composition

For each BCMC, the 16S rRNA V4 region was sequenced and analysed to determine the proportions that were „observed“. Escherichia and Pseudomonas were significantly lower than expected, and Bacillus and Corynebacterium were significantly higher than expected for BCMC1.

Effects of milk sample volume and cell count on bacterial composition estimates

Larger milk samples resulted in lower alpha diversity and fewer unexpected taxa assignments or bacteria that did not belong to the mock community. Most of these unexpected bacteria were Micrococcus or Tepidimonas.

Effects of the storage method on the detection of sham communities

Regardless of whether the cells were frozen in PBS or in water with 25 % v/v glycerol at -20 °C, the bacterial proportions of BCMC did not change.

Identification and tracking of bacteria

With the help of 16S rRNA gene sequence surveys, it is possible to identify and track bacterial populations. However, bias can occur at almost every stage of the process, from sampling to data analysis and interpretation. The most accurate method for automatic identification of bacteria consists of at least 10 grams of milk, mild cell lysis, proteinase K treatment and magnetic bead-based DNA purification.

Storage conditions of the collected cells and cell lysis method

The mock community remained unchanged despite different freeze-thaw cycles and storage conditions. It can be assumed that other upstream sample preparation steps have a greater influence than this one previously discovered. Although not tested directly, it has been previously demonstrated that liquid milk samples can be frozen directly when rapid cold transfer between collection sites is not possible.

Application of cell lysis and DNA extraction methods to assess the microbiota of raw and pasteurised milk

Although cell lysis techniques can bias the results of 16S rRNA gene amplicon DNA sequencing, they are unlikely to interfere with comparative microbiota studies of different milk samples. The variation within a sample caused by methodological adjustments was significantly less than the variation between samples. Microbial DNA extraction and analysis may require different methodological approaches for different types of cow’s milk samples.

Summary

Examining the microbiota of dairy products requires careful sample preparation and analysis to minimise bias and contamination problems. The use of a bacterial cell mock community (BCMC) can help to verify and improve the accuracy of results. When selecting cell lysis and DNA purification methods, milder techniques should be preferred to avoid bias. Storage conditions of the collected cells should also be carefully controlled to avoid changes in bacterial composition. The use of 16S rRNA gene sequencing can help identify and track bacterial populations, but possible biases must be considered.

miRNA as a biomarker for ALS – #JournalClub no.052

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miRNA extracted from extracellular vesicles is a robust biomarker of amyotrophic lateral sclerosis

Banack SA, Dunlop RA, Stommel EW, Mehta P, Cox PA. miRNA extracted from extracellular vesicles is a robust biomarker of amyotrophic lateral sclerosis. J Neurol Sci. 2022;442. doi:10.1016/j.jns.2022.120396

Promising Biomarker Found for Early-Stage ALS Diagnosis: miRNA Extracted from Extracellular Vesicles

Amyotrophic lateral sclerosis (ALS) affects tens of thousands of people worldwide, but diagnosing the disease accurately can be challenging. However, recent research has shown that miRNA extracted from extracellular vesicles (EVs) can be a reliable biomarker for early-stage ALS diagnosis. EVs are small, non-replicating particles released by cells that contain material from the source cell within their protective lipid bilayer membrane. This makes EVs a promising material for potential biomarkers, and researchers have discovered eight early-stage ALS-diagnostic miRNAs extracted from EVs enriched with L1CAM by immunoaffinity purification.

Internal Quality Control Measures Ensure Reproducibility of miRNA Biomarkers for Standard Clinical Use

However, for biomarkers to be useful in standard clinical use, they must be reproducible over time in different labs and with samples collected using different collection and storage protocols. The researchers have included internal quality control measures, such as melt-curves, no-template controls, and spike-in controls for RNA extraction and cDNA synthesis, to ensure the reproducibility of the miRNA biomarkers.

Utilizing Archived Samples from National ALS Biorepository for Reliable ALS Diagnosis

To obtain blood plasma samples for the study, archived samples from the U.S. National ALS Biorepository were utilized. The Biorepository is part of the U.S. National ALS Registry and is maintained by the Centers for Disease Control and Prevention and the Agency for Toxic Substances and Disease Registry. The blood plasma samples were collected from 50 patients with confirmed or probable ALS at different times and locations throughout the USA by phlebotomists. The recommended protocol included collecting the blood in K2 EDTA tubes and inverting the tubes ten times. The blood specimens were then packed in Styrofoam-insulated shippers with reusable cold-packs to maintain a temperature between 4 and 8 °C for overnight shipping to a central location for processing and storage.

Proper Collection and Storage of Blood Plasma Samples Crucial for ALS Biomarker Reproducibility

In the original study, the samples were processed within 1 hour of collection by being spun-down in a refrigerated centrifuge and then stored and transported at −80 °C. As controls were not available from the Biorepository, 50 samples of blood plasma from individuals without an ALS diagnosis (FDA Approval, #3003372368, Innovative Research Inc.) were used as the control population. The study was reviewed by Advarra IRB Pro00053269, and since de-identified participant data were used, the study was determined not to meet the DHHS definition of human subjects research under 45 CFR 46 and did not require IRB oversight.

Potential ALS Biomarkers Identified, But Further Research Required for Selectivity and Sensitivity Determination

The authors tested the generalizability of the biomarkers by using a cross-section of 50 ALS patients and found that 80% of the original biomarkers were statistically significant. However, larger sample sizes and comparisons with blood samples from ALS-mimic diseases are required to determine the biomarkers‘ selectivity and sensitivity.

Overall, the identification of eight miRNAs as potential biomarkers for ALS diagnosis using L1CAM for immunoaffinity purification and polymer-based precipitation for EV extraction is a significant development in the field. With more research, these biomarkers could become useful in the clinic for early-stage diagnosis and rate of progression prediction. Therefore, it is crucial to collect and store samples properly and follow preanalytic measures to ensure the biomarkers‘ reproducibility and reliability over time.

Prospective biobanking in the context of a low energy availability (LEA) study – #JournalClub no.049

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„Study protocol: prevalence of low energy availability and its relation to health and performance among female football players“

J. H. Rosenvinge et al., BMJ Open Sport Exerc. Med., Bd. 8, Nr. 1, S. e001219, Jan. 2022, doi: 10.1136/bmjsem-2021-001219.

LEA - Low energy availability in female elite athletes

Sustained low energy levels, or low energy availability (LEA), are associated with several potentially serious physiological and mental conditions. It has been published that LEA is prevalent in elite female endurance athletes and undermines the health and performance of female athletes.

Study protocol and storage of serum and plasma samples (prospective biobanking)

This paper describes a study protocol that addresses the prevalence, measurement, and correlates of LEA in relation to health and performance in female soccer players. In addition, serum and plasma samples from the subjects are stored in a biobank for future reference.

Four studies are conducted:

  1. to evaluate the accuracy of Global Positioning Systems (GPS)-based energy consumption monitoring devices using indirect calorimetry as the gold standard,
  2. to assess energy intake, quantify energy use, and investigate energy availability using self-report, dual-labeled water (DLW), and GPS monitoring devices,
  3. to determine the point prevalence of LEA with self-report, DLW, dual X-ray absorptiometry (DXA) to quantify muscle and bone mass distribution and density, and a series of hormone analyses; 
  4. and to investigate whether the prevalence of LEA varies over a full soccer season.

Scope of Study

Measurements of DXA and DLW are resource intensive and are collected from one professional soccer club (20 women). In contrast, the remaining data will be collected from four high profile soccer clubs competing in the Norwegian first or second division (60 women, ages 16-34) in Bergen. Participants will be required to provide blood samples for all hormonal data, which will be collected after an overnight fasting period (8-10 hours). A transvaginal ultrasound will be performed and examined in conjunction with serum testosterone, if needed, to look for possible symptoms of polycystic ovaries.