Sample Preservation Research Articles

InDrops-2: a flexible, versatile and cost-efficient droplet microfluidic approach for high-throughput scRNA-seq of fresh and preserved clinical samples.

The expansion of single-cell analytical techniques has empowered the exploration of diverse biological questions at the individual cells. Droplet-based single-cell RNA sequencing (scRNA-seq) methods have been particularly widely used due to their high-throughput capabilities and small reaction volumes. While commercial systems have contributed to the widespread adoption of droplet-based scRNA-seq, their relatively high cost limits the ability to profile large numbers of cells and samples. Moreover, as the scale of single-cell sequencing continues to expand, accommodating diverse workflows and cost-effective multi-biospecimen profiling becomes more critical. Herein, we present inDrops-2, an open-source scRNA-seq technology designed to profile live or preserved cells with a sensitivity matching that of state-of-the-art commercial systems but at a 6-fold lower cost. We demonstrate the flexibility of inDrops-2, by implementing two prominent scRNA-seq protocols, based on exponential and linear amplification of barcoded-complementary DNA, and provide useful insights into the advantages and disadvantages inherent to each approach. We applied inDrops-2 to simultaneously profile multiple human lung carcinoma samples that had been subjected to cell preservation, long-term storage and multiplexing to obtain a multiregional cellular profile of the tumor microenvironment. The scalability, sensitivity and cost efficiency make inDrops-2 stand out among other droplet-based scRNA-seq methods, ideal for large-scale studies on rare cell molecular signatures.

Effects of Different Voided Urine Sample Storage Time, Temperature, and Preservatives on Analysis with Multiplex Bead-Based Oncuria Bladder Cancer Immunoassay

Effects of Different Voided Urine Sample Storage Time, Temperature, and Preservatives on Analysis with Multiplex Bead-Based Oncuria Bladder Cancer Immunoassay

Witnessing a discrete microdroplet freezing event via real-time electrochemical monitoring of solution temperature.

Temperature monitoring has immediate relevance to many areas of research, from atmospheric environmental studies to biological sample and food preservation to chemical reactions. Here, we use a triple-barrel electrode to provide temperature readouts in bulk solution and microdroplets, as well as electrochemically monitor freezing events in a microdroplet. Using this method, we are able to identify distinct characteristics of a freezing aqueous droplet (supercooling, ice formation beginning and end, temperature change, and thawing) with greater temporal resolution than a standard thermocouple and without the use of microscopy. By correlating the amperometric signal change caused by alterations in the diffusion coefficient of the electrochemical system in response to temperature changes, we can calculate the instantaneous temperature at our electrode, as well as the physical behavior of ice formation and expansion. Our results suggest that these electrochemical techniques can provide real-time monitoring of the physical processes involved in aqueous temperature change and ice nucleation events. Here, we employ a novel technique using triple-barrel electrodes to provide temperature readouts in bulk solution and microdroplets, as well as electrochemically monitor freezing events in a microdroplet. Because ice nucleation spans many research fields, it is important to have a variety of tools that can be used to better understand these frozen systems. Our data shows that electrochemistry can provide real-time information on the thermal properties of aqueous environments, and these types of measurements can be extended to microdroplets. The electrochemical signal details all the significant moments in a droplet freezing event, allowing us to use electrochemistry as a stand-alone tool for monitoring freezing events with excellent temporal and spatial resolution.

An all-in-one microfluidic cryopreservation system and protocols with gradually increasing CPA concentration.

In regular biosample cryopreservation operations, dropwise pipetting and continuous swirling are ordinarily needed to prevent cell damage (e.g. sudden osmotic change, toxicity and dissolution heat) caused by the high-concentration cryoprotectant (CPA) addition process. The following CPA removal process after freezing and rewarming also requires multiple sample transfer processes and manual work. In order to optimize the cryopreservation process, especially for trace sample preservation, here we present a microfluidic approach integrating CPA addition, sample storage, CPA removal and sample resuspension processes on a 30 × 30 × 4 mm3 three-layer chip. The sample solution could be added into CPA solution with pre-generated increasing concentration to decrease possible osmotic damage. Utilizing specially designed microfluidic structure and fluid field analysis, on-chip sample enrichment and CPA removal were achieved. A novel dead-end micro valve strategy with a simplified control module was applied and evaluated to assist on-chip mixing and sample pellet resuspension. The entire biosample cryopreservation process was also performed that verified the functions of the integrated microfluidic platform. Altogether, this developed platform could be an effective approach to realize automatic, all-in-one, low-damage cryopreservation operation.

SnRNA-seq of long-preserved FFPE samples from colorectal liver metastasis lesions with diverse prognoses

Differences in prognostic outcomes are prevalent in patients with colorectal cancer liver metastases. Comparative analysis of tissue samples, particularly applying single-cell transcriptome sequencing technology, can provide a deeper understanding of potential impacting factors. However, long-term monitoring for prognosis determination necessitates extended preservation of tissue samples using formalin-fixed and paraffin-embedded (FFPE) treatments, which can cause substantial RNA degradation, presenting challenges to single-cell or single-nucleus sequencing. In this study, employing snRandom-seq, a single-nucleus RNA sequencing (snRNA-seq) technology specifically for FFPE samples, we tested multiple lesion samples from 18 distinctive colorectal cancer liver metastasis cases with diverse prognostic outcomes that have been preserved for at least three years (mostly over five years). The process yielded expression data from 82,285 cells. The high-quality snRNA-seq data demonstrate the feasibility of single-nucleus sequencing in long-term preserved FFPE samples, offering potential insights into the heterogeneity between different prognoses of colorectal cancer liver metastases, and the relationship between the heterogeneity within different lesions of the same patient and prognosis.

The use of 10% buffered formalin as a preservative agent when cerebrospinal fluid analysis is delayed.

To evaluate the utility of 10% buffered formalin in preserving canine cerebrospinal fluid samples when analysis was delayed. Inclusion criteria were dogs >10 kg having cerebrospinal fluid analysis performed as part of investigations at a referral hospital. Samples were submitted to an external laboratory in tubes containing Ethylenediaminetetraacetic acid (ETDA) as paired 0.5 mL samples; one with the addition of 0.05 mL of 10% buffered formalin and the other without. The samples were reviewed by a single pathologist who was blinded as to which sample contained formalin. Nucleated cell preservation was graded by the pathologist from 1 to 4 (1 being poor and 4 being excellent). Total protein was measured in both samples. Forty-five paired samples were included. There was no significant difference in detectability of nucleated cells between plain and formalin samples. Grade 3 was taken as the cut off for acceptable cell preservation. Based on all available samples and assessing the preservation of both nucleated cells and red blood cells, samples containing formalin were significantly more likely to be acceptably preserved. This preservation analysis was repeated on the 17 samples with at least one nucleated cell in both formalin and plain samples and was not statistically significant. The addition of formalin did not significantly improve the preservation of cerebrospinal fluid samples when analysis was delayed; however, concerns raised by previous authors regarding reduced cell preservation with addition of formalin were also not confirmed. Further large-scale studies are required to investigate the effect on nucleated cell preservation.

NMR metabolomics as a complementary tool to brix-acid tests for navel orange quality control of long-term cold storage.

Quality control plays a crucial role in maintaining the reputation of agricultural organizations by ensuring that their products meet the expected standards and preventing any loss during the packaging process. A significant responsibility of quality control is conducting periodic product assessments. However, subjective interpretation during physical inspections of fruits can lead to variability in reporting. To counter this, assessing total soluble solids (Brix) and percent acidity (Acid) can provide a more objective approach. Nevertheless, it is essential to note that many fruit metabolites can impact these parameters. Nuclear magnetic resonance (NMR) spectroscopy, particularly 1H-NMR, has become a popular tool for quality control in recent years due to its precision, sample preservation, and high throughput analysis. This manuscript investigates if the standard Brix/Acid tests are directly related to the levels of metabolites during cold storage. Using citrus as the model system, a metabolomics analysis was conducted to identify patterns in the cold storage metabolite profiles of the juice, albedo, and flavedo tissues. The results show that Brix (or total dissolved solids) correlates well with sucrose, glucose, and fructose levels and moderately with choline levels. Acid(percent acidity) levels displayed a negative correlation with both fructose and choline levels. Interestingly, the formate levels were susceptible to storage time and directly related to Acid measurements. This study suggests metabolomics could be a complementary technique to quality control of fruits in cold storage, especially with cost-effective desktop NMR spectrometers.

Are spinal cord and medulla samples from embalmed donors suitable for histological examination? A pilot study.

A general belief exists that tissue from anatomical donors, especially from the central nervous system (CNS), may not be suitable for histological investigation. This is based on the idea that fixation routinely used in embalming whilst optimal for enabling dissection, insufficiently preserves tissue architecture at the cellular level. However, anatomical donors represent a precious resource for microscopical investigation, provided that preservation is sufficient to enable recognition of structures at the histological level. This could considerably extend the biomedical knowledge currently gained from donations. In this study, we addressed this question and examined histologically, samples of medulla and spinal cord from donors from the Anatomical Gift Programme of RCSI in Dublin. Samples of medulla and spinal cord in the cervical, thoracic and lumbar regions were obtained with appropriate permissions from four different donors that had been previously embalmed for routine anatomical examination. The tissue obtained was processed for paraffin embedding and routine histology. Analysis revealed that several features were identifiable with good histological detail. A scoring system was applied to evaluate the level of histological preservation in the various samples by assessing the following parameters: tissue integrity, presence of leptomeninges along the entire section surface, distinction/adherence grey/white matter, identification of neurons, other cell nuclei, neuropil pattern, capillaries and other intraneural vessels. Lumbar spinal cord and to a lesser extent medulla and cervical spinal cord showed on average good quality of preservation. Conversely, thoracic spinal cord tended, in general, to be less well preserved. We can conclude that spinal cord and medulla samples from embalmed donors can be suitable for histological examination. These findings open new avenues of study of spinal cord and brain stem anatomy on cadaveric tissue from donors already existing in anatomy donation programmes. These results also pave the way for the potential development of new human models of study useful to spinal cord and brain stem researchers. Finally, this expands considerably the impact of the generosity of anatomical donors for the advancement of biomedical knowledge.

Benchmarking three DNA metabarcoding technologies for efficient detection of non-native cerambycid beetles in trapping collections

Individual sorting and identification of thousands of insects collected in mass trapping biosurveillance programmes is a labour-intensive and time-consuming process. Metabarcoding allows the simultaneous identification of multiple individuals in a single mixed sample and has the potential to expedite this process. However, detecting all the species present in a bulk sample can be challenging, especially when under-represented non-native specimens are intercepted. In this study, we quantified the effectiveness of DNA metabarcoding at detecting exotic species within six different mock communities of native and non-native species of European xylophagous cerambycid beetles. The main objective is to compare three different sequencing technologies (MinION, Illumina and IonTorrent) to evaluate which one is the most suitable in this context. Additionally, dry and wet (monopropylene glycol and water) collection methods were compared. Although not observing significant differences in the total number of species detected amongst the three sequencing technologies, the MinION detected a greater number of species in field-like samples. All three sequencing technologies achieved success in detecting and identifying closely-related species and species in low abundance. The capture method of insects in the field greatly influenced sample preservation and detection. Individuals captured in traps containing monopropylene and water had lower DNA concentration, leading to lower species detection rates compared to individuals killed using just an insecticide without any collection medium.

Volunteer Accuracy in a Benthic Macroinvertebrate Participatory Science Project

Concerns about the accuracy of volunteer-derived aquatic macroinvertebrate identifications and the resulting influence on calculated water quality metrics can limit use of volunteer-generated data in freshwater-focused participatory science programs. To address these concerns, we assessed 357 benthic macroinvertebrate quality control (QC) samples collected by volunteers using leaf packs, kick nets, and visual assessments between 2011 and 2016 for the Environmental Quality Institute (EQI) in North Carolina, USA. We reviewed each sample for quality, and compared macroinvertebrate identifications and water quality metric scores determined by volunteers to identification and metrics determined by an entomologist. About 80% of the QC samples were identified to be of high quality, indicating proper preparation, preservation, and labeling of samples. The majority of samples that received low quality ratings were improperly or poorly preserved. We recommend use of 95% (rather than 70%) ethanol for macroinvertebrate preservation, and increased communication to volunteers during QC sampling periods to enhance their success in properly preserving samples. We observed significant (p < 0.005) linear relationships between volunteer and entomologist-derived water quality metrics including a biotic index, Taxa Richness, and percent intolerant Ephemeroptera, Plecoptera and Trichoptera (EPT) taxa. However, visual assessments – those in which no sampling equipment was used to collect aquatic macroinvertebrates – reduced the goodness of fit between volunteer and entomologist-derived biotic index scores and artificially increased the goodness of fit between volunteer and entomologist derived Taxa Richness and percent EPT scores. We recommend calculating water quality metrics based only on leaf pack and kick net samples collected by volunteers.

Practical considerations for plunge freezing samples over 40 °C for Cryo-EM

Cryo-EM is now an established tool for examining samples in their native, hydrated states—a leap made possible by vitrification. Utilising this sample preparation method to directly visualise temperature-responsive samples allows for deeper insights into their structural behaviours under functional conditions. This requires samples to be plunge-frozen at elevated temperatures and presents additional challenges, including condensation within the blotting chamber and difficulties in maintaining a stable sample temperatures. Here, we address these challenges and suggest practical strategies to minimise condensation and reduce temperature fluctuations during the plunge-freezing of samples at elevated temperatures (>40 °C). By preheating equipment and reducing chamber humidity and blotting times, we can improve sample preservation and grid reproducibility. These considerations are then demonstrated on poly(N-isopropylacrylamide) microgels, which exhibit a volume phase transition due to temperature changes.

On the generation of light hydrocarbons from the closed pores of Jurassic strata, Sichuan Basin

Light hydrocarbons (C1-C9) offer a specific opportunity to study petroleum generation mechanisms. However, a significant amount of light hydrocarbons evaporate during sample collection, preservation, and preparation. This study aims to identify the origin of light hydrocarbons preserved in the closed pores of Jurassic shale strata from the Sichuan Basin where evaporative losses have been minimized. Twenty-one samples of various maturity levels and lithofacies were investigated using a new approach based on online decrepitation-gas chromatography. Light hydrocarbons were released from the closed pores of finely stratified mudstone, limestone, and siltstone. Among these, gaseous hydrocarbons in the C1-C5 range were utilized to discriminate between petroleum generated by two opposing reactions, i.e., free radical and carbenium ion cracking. Accordingly, in situ petroleum can be divided into three types. (1) Free radical cracking type: a predominance of methane and straight-chain alkanes in the gaseous hydrocarbon range; (2) Carbenium ion cracking type: a deficiency of methane and straight-chain alkanes in gaseous hydrocarbons; (3) Mixed cracking type: their gaseous hydrocarbons exhibit intermediate compositions between those of free radical and carbenium ion cracking types. Differences in the chemical composition of the gaseous hydrocarbons, which can be used to discriminate between the two generation mechanisms, were further supported by variations in the mineral composition, e.g., calcite and mixed-layer clay minerals are characteristic of free radical reaction and carbenium ion reaction, respectively. The present classification diagrams for gaseous hydrocarbons are based on the Jurassic shale strata of the Sichuan Basin. This novel approach for determining the whole range of hydrocarbons from closed pores shows promising prospects for deciphering the origin of light hydrocarbons and thus may be extended to other regions of interest.

QLTI-09. CLINICAL TRIALS IN NEURO-ONCOLOGY: DESIGN, LOGISTICS, CHALLENGES, AND PRACTICAL CONSIDERATIONS

Abstract Timely, secure and reliable transportation, precise handling and storage of biological specimens are critical for the success of clinical research. Research in neuro-oncology often involves complex assays such as next-generation sequencing where the integrityof brain tumor specimens is paramount. These challenges are pronounced in most Lower Middle-Income Countries (LMIC), due to climatic factors and infrastructure limitations, necessitating stringent measures to preserve sample quality and their genetic data integrity for downstream analysis. In this study, we conducted extensive cohort analysis on 80 tissues randomly picked samples of the 3600 samples collected across India to assess the impact of collection protocols, transportation logistics, and long-term storage on the genetic material of tissues. The quality and quantity of the isolated nucleic acids was determined by BioAnalyzer and Qubit parameters. Tissue samples were collected and stored at -80°C in RNAstable® within 4-12 hrs of collection. The DNA and RNA obtained from tissue samples stored for over one year was found to be of good quality with corresponding DNA Integrity Number (DIN) and RNA Integrity Number (RIN)between 7-9. Also, optimum quantity of DNA and RNA was obtained for whole exome and transcriptome sequencing. Upon sequencing, these samples showed good read coverage of 100X or more. These findings provide critical insights into the measures necessary for optimizing sample preservation and transportation to ensure accurate research outcomes. The results indicate that appropriate sample collection, transportation, and long-term storage of bio-specimens can be used effectively for future research. Additionally, the outcome underscores the potential for long-term studies in neuro-oncology using archived biological samples without compromising data integrity.

An outbreak of Clostridium perfringens infection on a training ship anchored in Busan, Korea.

In September 2023, an outbreak of food-borne disease occurred among students on a training ship docked in Busan. This was an epidemiological investigation with the aim of improving infection prevention activities and group meal service practices on board ships. In this study, a case was defined as an individual who experienced diarrhea more than twice a day during their training period aboard the training ship. A total of 171 exposed individuals including 6 food handlers was well-defined; therefore, a retrospective cohort study was conducted. We administered a questionnaire and conducted laboratory tests including 38 rectal swab samples. Relative risk (95% confidence interval) for each food item was calculated. Of the 165 students and school staff members, 41 met the case definition, resulting in an attack rate of 24.8%; all cases were students. The shape of the epidemic curve was unimodal, with the peak from 00:00 to 06:00 on September 7, 2023. Clostridium perfringens was detected in 9 cases, and no other pathogens were found. Significant relative risk was shown in 11 different food items. C. perfringens was the causative pathogen of this outbreak on the training ship. Due to the lack of preserved food samples, the exact source of infection could not be confirmed. Ships are not classified as collective dining facilities, leaving them in a management blind spot. Therefore, specialized guidelines, voluntary inspections by the operating entities, and continuous education for managers and staff are necessary.

Preservation of Aquatic Environmental DNA Using Cationic Detergents

ABSTRACTEnvironmental DNA (eDNA) analysis is a powerful tool for quantifying and assessing the diversity of organisms in the environment. Unfortunately, isolating eDNA from aquatic environments is challenging due to the difficulties associated with water collection, preservation of samples during transportation, and onsite filtration. These processes are expensive and time‐consuming and can lead to eDNA degradation. These difficulties can be addressed by preserving eDNA in the collected water. In this study, we assessed the effect of short‐ and long‐term water storage using three different cationic surfactants on the half‐life of zebrafish (Danio rerio) mitochondrial DNA (mtDNA) in mesocosm water. The surfactants used were benzalkonium chloride (BAC), cetylpyridinium chloride (CPC), and cetyltrimethylammonium bromide (CTAB). We observed that CPC and CTAB treatment extended the half‐life of mtDNA by 3–5 times. Analysis by quantitative polymerase chain reaction (qPCR) demonstrated a mtDNA retention rate of 17.6%, 26.3%, and 2.2% for CPC, CTAB, and BAC, respectively, compared to 0.1% in untreated water after 30 days. The preservation of mtDNA by cationic surfactants was attributed to their bactericidal and cytotoxic properties as well as their electrostatic interaction with DNA molecules, as observed by spectrofluorometric analysis and subsequent precipitation. Our results demonstrated an inexpensive and convenient method to protect eDNA in water and improve its extraction.

Prevalence of Human Papillomavirus-Associated Head and Neck Cancer in Rwanda: A 10-Year Review.

Head and neck cancer (HNC) is a significant global health burden, with late presentation leading to complex treatment. While human papillomavirus (HPV) infection has been implicated in HNC, data from low- and middle-income countries (LMICs) are limited. In this study, we investigated the prevalence and role of HPV in head and neck cancers diagnosed in Rwanda. A retrospective cross-sectional study was conducted using Rwanda Cancer Registry from January 2011 through December 2020. p16 immunohistochemistry as a surrogate for HPV was performed on a randomly selected case. p16-positive cases were genotyped. A total of 1001 patients with HNC were identified; 82% (n = 819) had squamous cell carcinoma. The mean age at diagnosis was 51.1 years, with a majority being males (58%). Oral cavity and lip (27%) were the most common primary cancer sites. Stage was unknown in most cases (75%, n = 747). HIV status was known in 33% (n = 334) of patients with 10% (n = 33) HIV-positive; 22% of 202 randomly selected cases were p16-positive; 34% of the p16-positive cases were oropharynx. PCR analysis of p16-positive cases showed 19% HPV positivity, and HPV16 was the most common high-risk HPV strain, and 55.5% were recorded HPV-positive by PCR. HNC cases in Rwanda have been increasing from 2011 to 2020, with a significant portion being HPV-positive. Strategies to implement routine testing for p16, especially in oropharynx cancer patients, improved preservation of tissue samples, collection of comprehensive information including cancer risk factors, staging, and treatment are needed in Rwanda.

Diagenetic History and Biosignature Preservation Potential of Fine‐Grained Rocks at Hogwallow Flats, Jezero Crater, Mars

AbstractThe Mars 2020 Perseverance rover discovered fine‐grained clastic sedimentary rocks in the “Hogwallow Flats” member of the “Shenandoah” formation at Jezero crater, Mars. The Hogwallow Flats member shows evidence of multiple phases of diagenesis including Fe/Mg‐sulfate‐rich (20–30 wt. %) outcrop transitioning downward into red‐purple‐gray mottled outcrop, Fe/Mg clay minerals and oxides, putative concretions, occasional Ca sulfate‐filled fractures, and variable redox state over small (cm) spatial scales. This work uses Mastcam‐Z and SuperCam instrument data to characterize and interpret the sedimentary facies, mineralogy and diagenetic features of the Hogwallow Flats member. The lateral continuity of bedrock similar in tone and morphology to Hogwallow Flats that occurs over several km within the western Jezero sedimentary fan suggests widespread deposition in a lacustrine or alluvial floodplain setting. Following deposition, sediments interacted with multiple fluids of variable redox state and salinity under habitable conditions. Three drilled sample cores were collected from this interval of the Shenandoah formation as part of the Mars Sample Return campaign. These samples have very high potential to preserve organic compounds and biosignatures. Drill cores may partially include dark‐toned mottled outcrop that lies directly below light‐toned, sulfate‐cemented outcrop. This facies may represent some of the least oxidized material observed at this interval of the Shenandoah formation. This work reconstructs the diagenetic history of the Hogwallow Flats member and discusses implications for biosignature preservation in rock samples for possible return to Earth.

From sampling to cellblock: The fully automated journey of cytological specimens.

In recent years, technological innovation have emerged to standardize pathology laboratory processes and reduce the handling of diagnostic samples. Among them is an automatic tissue embedding system that eliminates the need for manual activity in tissue paraffin embedding, thereby improving sample preservation. Unfortunately, this system cannot be used for cytological specimens due to the lack of an effective holder to support the procedure steps. In this study, we evaluated the performance of a commercial polymer matrix to enable and standardize the automatic paraffin embedding of cytological material from different organs and sources. Cytological samples from 40 patients were collected on the matrices and submitted for fully automatic workflow preparation, from formalin fixation until paraffin block, using the Sakura embedding system. Our results demonstrated the feasibility of the automated procedure, from loading cytological sample onto the matrix to obtaining the paraffin cellblock, thereby avoiding manual manipulation of cellular material. All samples resulted adequately processed and paraffin-embedded showing satisfactory tissue permeation by processing reagents, optimal preservation of cytoplasmic and nuclear details, and good quality of staining results on paraffin sections. Automated embedding of cytological samples eliminates the risk of lost specimens, reduces laboratory burden, standardizes procedures, increases diagnostic yield, and ultimately improves patients' management.

Mercury speciation in environmental samples associated with artisanal small-scale gold mines using a novel solid-phase extraction approach to sample collection and preservation

In artisanal small-scale gold mines (ASGM), mercury (Hg) is known to pollute nearby river waters and sediments where it can be methylated to the highly bioavailable methylmercury (MeHg). The assessment of Hg speciation in water samples has been challenging for many years, with recommended procedures often not adequately allowing for analysis of samples in a suitable timeframe. Using a novel solid-phase extraction (SPE) method for sampling and preservation of Hg species, representative speciation data can be safely and easily collected and retained for up to 4-weeks (MeHg = 115 ± 8% refrigerated and 109 ± 13% unrefrigerated storage; Hg2+ = 100 ± 14% refrigerated and 94 ± 12% unrefrigerated storage). Concentrations of MeHg in environmental water samples and drinking water were below detection limit across two ASGM sites in western Kenya and concentrations of Hg2+ were below drinking water guidelines; however, drinking water sources contribute 20–30% of the tolerable weekly intake of Hg, indicating a need to minimise exposure of Hg from dietary sources to prevent Hg poisoning. Sediments from receiving rivers at ASGM sites showed total Hg concentrations above guideline limits (0.08–1.84 mg kg−1 total Hg) along the length of the river; however, MeHg concentrations fluctuated dependent on the stagnation of the river due to damns and ponds (5.9 ± 14.3 µg kg−1 MeHg). The findings show that SPE can be used as a robust sample collection and preservation approach for Hg speciation, which can better inform mitigation measures, understand ecological and human health implications, and improve environmental monitoring.

Submissions of diagnostic samples of two critically endangered species of handfish (Branchionichthys hirsutus and Thymichthys politus) from a public aquarium from 2018 to 2024.

Handfish (Family Brachionichthyidae) is the most threatened marine teleost fish family, however, there is little information on handfish health. We reviewed the results of submissions of mortalities from captive and captive bred spotted handfish (Branchionichthys hirsutus (Lacepède, 1804)) and red handfish (Thymichthys politus (Richardson, 1844)) from a public aquarium from January 2018 to February 2024. Seventeen cases for spotted handfish (comprising 33 individuals) and five cases for red handfish (one individual each) were submitted for mortality investigation. In 2018-2019, six of seven cases were diagnosed with scuticociliatosis. Other conditions included epitheliocystis (1 of 17), infections with metazoan parasites (2 of 17 for spotted handfish and 2 of 5 for red handfish) and hepatic lipidosis (4 of 17). Most submissions were fixed samples for histology with only one fish suitable for microbiology. We recommend development of a coordinated health program for all captive breeders and aquaria, which should include sampling protocols, collection, and preservation in a range of fixatives of all the dying or dead handfish and adapting sublethal sampling, including tank water to assess presence of pathogens and other microorganisms. Risk factors for handfish health in captivity should be assessed. Handfish health database should be established to avoid loss of corporate knowledge in this area.