Storage Of Specimens Research Articles

Impact of sample processing delays on plasma markers of inflammation, chemotaxis, cell death, and blood coagulation.

Biosampling studies in critically ill patients traditionally involve bedside collection of samples followed by local processing (ie. centrifugation, aliquotting, and freezing) and storage. However, community hospitals, which care for the majority of Canadian patients, often lack the infrastructure for local processing and storage of specimens. A potential solution is a "simplified" biosampling protocol whereby blood samples are collected at the bedside and then shipped to a central site for processing and storage. One potential limitation of this approach is that delayed processing may alter sample characteristics. To determine whether delays in blood sample processing affect the stability of cytokines (IL-6, TNF, IL-10, IFN-γ), chemokines (IL-8, IP-10, MCP-1, MCP-4, MIP-1α, MIP-1β), cell-free DNA (cfDNA) (released by dying cells), and blood clotting potential in human blood samples. Venous blood was collected into EDTA and citrate sample tubes and stored at room temperature (RT) or 4°C for progressive intervals up to 72 hours, prior to processing. Plasma cytokines and chemokines were quantified using single or multiplex immunoassays. cfDNA was measured using Picogreen DNA Quantification. Blood clotting potential was measured using a thrombin generation assay. Blood samples were collected from 9 intensive care unit (ICU) patients and 7 healthy volunteers. Admission diagnoses for the ICU patients included sepsis, trauma, ruptured abdominal aortic aneurysm, intracranial hemorrhage, gastrointestinal bleed, and hyperkalemia. After pre-processing delays of up to 72 hours at RT or 4°C, no significant changes were observed in plasma cytokines, chemokines, cfDNA, or thrombin formation. Delayed sample processing for up to 72 hours at either RT or 4°C did not significantly affect cytokines, chemokines, cfDNA, or blood clotting potential in plasma samples from healthy volunteers and ICU patients. A "simplified" biosampling protocol is a feasible solution for conducting biosampling research at hospitals without local processing capacity.

The Environmental Impact of a High-Altitude Medical Research Expedition.

Joyce, Kelsey E., Catherine A. Campbell, Arthur R. Bradwell, Samuel J.E. Lucas, Christopher T. Lewis, Rebekah A.I. Lucas, and Mark Edsell. The environmental impact of a high-altitude medical research expedition. High Alt Med Biol. 00:00-00, 2024. Introduction: Scientists must begin examining the environmental cost(s) of their research. The purpose of this study was to evaluate a component of the environmental impact of a high-altitude medical research expedition by totaling the carbon dioxide (CO2) emissions calculated from as many direct and indirect sources as possible. Methods: Eighteen individuals flew from London to Bagdogra (via Delhi), and then drove onward to Lachung (via Gangtok) where they began their ascent on foot to 4,800 m (Kanchenjunga National Park, Sikkim). Several research experiments were conducted throughout the expedition, which required use of a laboratory centrifuge, solid CO2 (specimen storage), rechargeable laptop computers and battery-powered oximeters. International Civil Aviation Organization calculators estimated aviation CO2 production. Land emissions were calculated for Mahindra vehicles. Solid waste was weighed and CO2 emissions estimated for its incineration. Results: Total CO2 emissions equated to ∼16.7 tonnes from the following sources: air and land transportation of expedition team (87.3%); sublimation and transportation of solid CO2 (7.7%), waste incineration (0.58%), generator transportation and gasoline (12 l) combustion (0.48%), and battery transportation (3.3%). Conclusions: Air travel contributed the most to the overall environmental cost of the research expedition. Further investigation is required to contextualize these findings in relation to lab-based alternative(s).

Early unrecognised SARS-CoV-2 introductions shaped the first pandemic wave, Sweden, 2020.

BackgroundDespite the unprecedented measures implemented globally in early 2020 to prevent the spread of SARS-CoV-2, Sweden, as many other countries, experienced a severe first wave during the COVID-19 pandemic.AimWe investigated the introduction and spread of SARS-CoV-2 into Sweden.MethodsWe analysed stored respiratory specimens (n = 1,979), sampled 7 February-2 April 2020, by PCR for SARS-CoV-2 and sequenced PCR-positive specimens. Sequences generated from newly detected cases and stored positive specimens February-June 2020 (n = 954) were combined with sequences (Sweden: n = 730; other countries: n = 129,913) retrieved from other sources for Nextstrain clade assignment and phylogenetic analyses.ResultsTwelve previously unrecognised SARS-CoV-2 cases were identified: the earliest was sampled on 3 March, 1 week before recognised community transmission. We showed an early influx of clades 20A and 20B from Italy (201/328, 61% of cases exposed abroad) and clades 19A and 20C from Austria (61/328, 19%). Clade 20C dominated the first wave (20C: 908/1,684, 54%; 20B: 438/1,684, 26%; 20A: 263/1,684, 16%), and 800 of 1,684 (48%) Swedish sequences formed a country-specific 20C cluster defined by a spike mutation (G24368T). At the regional level, the proportion of clade 20C sequences correlated with an earlier weighted mean date of COVID-19 deaths.ConclusionCommunity transmission in Sweden started when mitigation efforts still focused on preventing influx. This created a transmission advantage for clade 20C, likely introduced from ongoing cryptic spread in Austria. Therefore, pandemic preparedness should have a comprehensive approach, including capacity for large-scale diagnostics to allow early detection of travel-related cases and community transmission.

Increasing power in screening trials by testing control-arm specimens: application to multicancer detection screening.

Cancer screening trials have required large sample sizes and long time-horizons to demonstrate cancer mortality reductions, the primary goal of cancer screening. We examine assumptions and potential power gains from exploiting information from testing control-arm specimens, which we call the "intended effect" (IE) analysis that we explain in detail herein. The IE analysis is particularly suited to tests that can be conducted on stored specimens in the control arm, such as stored blood for multicancer detection (MCD) tests. We simulated hypothetical MCD screening trials to compare power and sample size for the standard vs IE analysis. Under two assumptions that we detail herein, we projected the IE analysis for 3 existing screening trials (National Lung Screening Trial [NLST], Minnesota Colon Cancer Control Study [MINN-FOBT-A], and Prostate, Lung, Colorectal, Ovarian Cancer Screening Trial-colorectal component [PLCO-CRC]). Compared with the standard analysis for the 3 existing trials, the IE design could have reduced cancer-specific mortality P values 6-fold (NLST), 33-fold (MINN-FOBT-A), or 260 000-fold (PLCO-CRC) or, alternately, reduced sample size (90% power) by 25% (NLST), 47% (MINN-FOBT-A), or 63% (PLCO-CRC). For potential MCD trial designs requiring 100 000 subjects per arm to achieve 90% power for multicancer mortality for the standard analysis, the IE analysis achieves 90% power for only 37 500-50 000 per arm, depending on assumptions concerning control-arm test-positives. Testing stored specimens in the control arm of screening trials to conduct the IE analysis could substantially increase power to reduce sample size or accelerate trials and could provide particularly strong power gains for MCD tests.

Evaluation of the feasibility and efficacy of point-of-care antibody tests for biomarker guided management of COVID-19.

Biomarker guided therapy could improve management of COVID-19 inpatients. Although some results indicate that antibody tests are prognostic, little is known about patient management using point-of-care (POC) antibody tests. COVID-19 inpatients were recruited to evaluate 2 POC tests: LumiraDX and RightSign. Ease of use data was collected. Blood was also collected for centralized testing using established antibody assays (GenScript cPass). A nested case-control study assessed if POC tests conducted on stored specimens were predictive of time to sustained recovery, mortality, and a composite safety outcome. While both POC tests exhibited moderate agreement with the GenScript assay (both agreeing with 89% of antibody determinations), they were significantly different from the GenScript assay. Treating the GenScript assay as the gold standard, the LumiraDX assay had 99.5% sensitivity and 58.1% specificity while the RightSign assay had 89.5% sensitivity and 84.0% specificity. The LumiraDX assay frequently gave indeterminant results. Both tests were significantly associated with clinical outcomes. Although both POC tests deviated moderately from the GenScript assay, they predicted outcomes of interest. The RightSign test was easier to use and was more likely to detect those lacking antibody compared to the LumiraDX test treating GenScript as the gold standard.

A multi-center evaluation of a novel IVF cryostorage device in an active clinical setting

The objective of this study was to evaluate the function, and usability of a novel automated software-guided cryostorage system in an active IVF laboratory setting. The investigational device (ID) was installed at 3 IVF laboratories (sites: α, β, and γ). A total of 15 embryologists were trained to use the ID. Mock patient specimens containing mirrored live patient data were handled using the ID. Temperature readings were recorded every minute. Successful identification, storage, and retrieval of mock patient specimens by the ID were evaluated. To assess an LN2 pressure builder, the frequency of use and events of workflow interruption were logged. Student’s t-test was used to determine statistical significance. The ID was in active use for 164 days total. During this time, 329 mock patient egg and embryo cohorts were handled by the ID. The mean ± SD temperatures during active use were: α, − 176.57 ± 1.83 °C; β, − 178.21 ± 2.75 °C; γ, − 178.98 ± 1.74 and did not differ significantly. The highest recorded temperatures were: α, − 165.14 °C; β, − 157.41 °C; γ, − 164.45 °C. A total of 1064 automation transactions on 409 specimen vessels were performed. Data was managed on 1501 eggs and embryos. The ID did not lose or misplace any specimen data or vessels, and no mock specimen was exposed to a detrimental (> − 150 °C) temperature excursion. Over the 25 LN2 pressure builder usages during 99 total days, there was 1 occurrence where usage interrupted workflow due to a lack of LN2 pressure. The ID has advantages over the current manual-based cryostorage systems, including radio frequency identification (RFID) tracking, automation of manual tasks, and software guidance to ensure accurate specimen storage and retrieval. The results of this study indicate that the ID can be integrated into active IVF laboratories.

PrecivityAD2™ Blood Test: Analytical Validation of an LC-MS/MS Assay for Quantifying Plasma Phospho-tau217 and Non-Phospho-tau217 Peptide Concentrations That Are Used with Plasma Amyloid-β42/40 in a Multianalyte Assay with Algorithmic Analysis for Detecting Brain Amyloid Pathology.

Alzheimer's disease (AD) is a progressive irreversible neurodegenerative disorder that represents a major global public health concern. Traditionally, AD is diagnosed using cerebrospinal fluid biomarker analysis or brain imaging modalities. Recently, less burdensome, more widely available blood biomarker (BBM) assays for amyloid-beta (Aβ42/40) and phosphorylated-tau concentrations have been found to accurately identify the presence/absence of brain amyloid plaques and tau tangles and have helped to streamline AD diagnosis. However, few BBMs have been rigorously analytically validated. Herein, we report the analytical validation of a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) multiplex method for quantifying plasma phosphorylated-tau217 (p-tau217) and non-phosphorylated-tau217 (np-tau217) peptide concentrations. We combined the p-tau217/np-tau217 concentrations ratio (%p-tau217) and the previously validated LC-MS/MS multiplex assay for plasma Aβ42/40 into a new multianalyte assay with algorithmic analysis (MAAA; PrecivityAD2™ test) that identifies brain amyloid status based on brain amyloid positron emission tomography. We found (a) the %p-tau217 assay is precise, accurate, sensitive, and linear over a wide analytical measurement range, and free from carryover and interference; (b) the pre-analytical specimen collection, processing, storage, and shipping conditions that maintain plasma tau peptide stability; and (c) using the measured analytical imprecision for plasma Aβ42/40 and p-tau217/np-tau217 levels in a worst-case scenario model, the PrecivityAD2 test algorithm for amyloid pathology classification changed for only 3.5% of participants from brain amyloid positive to negative, or from negative to positive. The plasma sample preparation and LC-MS/MS methods underlying the PrecivityAD2 test are suitable for use in the clinical laboratory and valid for the test's intended purpose: to aid in the diagnostic evaluation of individuals aged 55 and older with signs or symptoms of mild cognitive impairment or dementia.

Direct immunofluorescence demystified: Essential insights and recent advances for dermatologists.

Direct immunofluorescence (DIF) is widely used in dermatopathology for the diagnosis of autoimmune blistering diseases (AIBDs), cutaneous vasculitis, and connective tissue disorders. Although it is easy and useful to perform, it needs technical expertise and experience for proper interpretation. The yield of DIF depends on multiple factors including the adequacy, transportation, storage, processing, and interpretation of the biopsy specimen. Effective collaboration between the dermatologist and dermatopathologist along with meticulous clinico-pathological correlation is crucial for accurately interpreting DIF in the appropriate clinical context. In this narrative review of DIF in dermatology, we discuss the indications of DIF, recent updates on the selection of optimum biopsy sites, basic techniques of DIF including the classical transport medium and its alternatives, processing and staining technique, patterns in various diseases, advancements such as serration pattern analysis, and latest recommendations on the use of DIF in cutaneous disorders.

Metabolism study of 3-chloromethcathinone (3-CMC) by dried blood spot (DBS) sampling after controlled administration using a murine model.

The metabolism of 3-chloromethcathinone (3-CMC) was studied after controlled administration in a murine model using the dried blood spot (DBS) technique for the sampling, storage and purification of blood samples. Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) was used for the identification of metabolites and investigation of their fragmentation pattern. Subsequently, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for their identification and 3-CMC quantification in routine workload. The main metabolites identified were two stereoisomers of dihydro-CMC, N-demethyl-CMC, and dihydro-N-demethyl-CMC. The stability of 3-CMC and of its metabolites deposited on DBS was evaluated by replicate analyses after 30, 50, and 90 days, demonstrating a decrease in concentration. It was more pronounced for 3-CMC, with -67% and -82% percentage deviation from the initial concentrations, and for N-demethyl 3-CMC (decrease comprised between -48% and -88%) than for the di-hydro metabolites, ranging from -5% to -37%. Regardless, all of them were detectable till 90 days after deposition as DBS. The possibility of identifying 3-CMC and its metabolites with high sensitivity is an invaluable tool for the diagnosis of exposure to the substance, also in low doses or after some hours, and for various applications in clinical and forensic toxicology, such as driving under the influence, drug-facilitated crimes, and addiction to intoxications. DBS demonstrated to be a reliable technique for the sampling, storage, and purification of the blood specimen for 3-CMC and metabolite detection.

Changes in Alzheimer Disease Blood Biomarkers and Associations With Incident All-Cause Dementia

Plasma biomarkers show promise for identifying Alzheimer disease (AD) neuropathology and neurodegeneration, but additional examination among diverse populations and throughout the life course is needed. To assess temporal plasma biomarker changes and their association with all-cause dementia, overall and among subgroups of community-dwelling adults. In 1525 participants from the US-based Atherosclerosis Risk in Communities (ARIC) study, plasma biomarkers were measured using stored specimens collected in midlife (1993-1995, mean age 58.3 years) and late life (2011-2013, mean age 76.0 years; followed up to 2016-2019, mean age 80.7 years). Midlife risk factors (hypertension, diabetes, lipids, coronary heart disease, cigarette use, and physical activity) were assessed for their associations with change in plasma biomarkers over time. The associations of biomarkers with incident all-cause dementia were evaluated in a subpopulation (n = 1339) who were dementia-free in 2011-2013 and had biomarker measurements in 1993-1995 and 2011-2013. Plasma biomarkers of amyloid-β 42 to amyloid-β 40 (Aβ42:Aβ40) ratio, phosphorylated tau at threonine 181 (p-tau181), neurofilament light (NfL), and glial fibrillary acidic protein (GFAP) were measured using the Quanterix Simoa platform. Incident all-cause dementia was ascertained from January 1, 2012, through December 31, 2019, from neuropsychological assessments, semiannual participant or informant contact, and medical record surveillance. Among 1525 participants (mean age, 58.3 [SD, 5.1] years), 914 participants (59.9%) were women, and 394 participants (25.8%) were Black. A total of 252 participants (16.5%) developed dementia. Decreasing Aβ42:Aβ40 ratio and increasing p-tau181, NfL, and GFAP were observed from midlife to late life, with more rapid biomarker changes among participants carrying the apolipoprotein E epsilon 4 (APOEε4) allele. Midlife hypertension was associated with a 0.15-SD faster NfL increase and a 0.08-SD faster GFAP increase per decade; estimates for midlife diabetes were a 0.11-SD faster for NfL and 0.15-SD faster for GFAP. Only AD-specific biomarkers in midlife demonstrated long-term associations with late-life dementia (hazard ratio per SD lower Aβ42:Aβ40 ratio, 1.11; 95% CI, 1.02-1.21; per SD higher p-tau181, 1.15; 95% CI, 1.06-1.25). All plasma biomarkers in late life had statistically significant associations with late-life dementia, with NfL demonstrating the largest association (1.92; 95% CI, 1.72-2.14). Plasma biomarkers of AD neuropathology, neuronal injury, and astrogliosis increase with age and are associated with known dementia risk factors. AD-specific biomarkers' association with dementia starts in midlife whereas late-life measures of AD, neuronal injury, and astrogliosis biomarkers are all associated with dementia.

Prevalence of hepatitis A virus among migrant workers in Qatar: A national study.

Hepatitis A virus (HAV) is the predominant cause of acute viral hepatitis worldwide; however, data on HAV antibody prevalence (seroprevalence) among migrant populations are limited. This study aimed to investigate HAV seroprevalence among Qatar's migrant craft and manual workers (CMWs), constituting approximately 60% of the country's population. HAV antibody testing was conducted on stored serum specimens obtained from CMWs during a nationwide severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) population-based cross-sectional survey between July 26 and September 9, 2020. Associations with HAV infection were investigated through regression analyses. Of the 2,607 specimens with HAV antibody test results, 2,393 were positive, and 214 were negative. The estimated HAV seroprevalence among CMWs was 92.0% (95% CI: 90.9-93.1%). HAV seroprevalence was generally high but exhibited some variation, ranging from 70.9% (95% CI: 62.4-78.2%) among Sri Lankans to 99.8% (95% CI: 98.2-99.9%) among Pakistanis. The multivariable regression analysis identified age, nationality, and educational attainment as statistically significant factors associated with HAV infection. Relative to CMWs aged ≤29 years, the adjusted relative risk (ARR) was 1.06 (95% CI: 1.03-1.10) in CMWs aged 30-39 years and reached 1.15 (95% CI: 1.10-1.19) in those aged ≥50 years. In comparison to Indians, the ARR was lower among Sri Lankans, assessed at 0.81 (95% CI: 0.72-0.91), but higher among Nepalese at 1.07 (95% CI: 1.04-1.11), Bangladeshis at 1.10 (95% CI: 1.07-1.13), Pakistanis at 1.12 (95% CI: 1.09-1.15), and Egyptians at 1.15 (95% CI: 1.08-1.23). No evidence for differences was found by geographic location or occupation. HAV seroprevalence among Qatar's CMW population is very high, with over nine out of every ten individuals having been exposed to this infection, likely during childhood.

A case-cohort study of per- and polyfluoroalkyl substance concentrations and incident prostate cancer in the cancer prevention Study-II LifeLink cohort study

IntroductionPer- and polyfluoroalkyl substances (PFAS) are environmentally persistent, potentially carcinogenic chemicals. Previous studies investigating PFAS exposure and prostate cancer yielded mixed findings. We aimed to investigate associations between PFAS exposure and incident prostate cancer in a large cohort of U.S. men, overall and by selected demographic, lifestyle, and medical-related characteristics. MethodsWe conducted a case-cohort study among Cancer Prevention Study-II LifeLink Cohort participants who, at baseline (1998–2001), had serum specimens collected and no prior cancer diagnosis. The study included all men diagnosed with prostate cancer (n = 1610) during follow-up (baseline–June 30, 2015) and a random sub-cohort of 500 men. PFAS concentrations [perfluorohexane sulfonic acid (PFHxS), perfluorooctane sulfonate (PFOS), perfluorononanoic acid (PFNA), and perfluorooctanoic acid (PFOA)] were measured in stored serum specimens. We used multivariable Cox proportional hazards models to estimate associations between PFAS concentrations and prostate cancer, overall and by selected characteristics (grade, stage, family history, age, education, smoking status, and alcohol consumption). ResultsProstate cancer hazards were slightly higher among men with concentrations in the highest (Q4) vs lowest quartile (Q1) for PFHxS [hazard ratio (HR) (95% CI): 1.18 (0.88–1.59)] and PFOS [HR (95% CI): 1.18 (0.89–1.58)], but not for PFNA or PFOA. However, we observed heterogeneous associations by age, family history of prostate cancer (PFHxS), alcohol consumption (PFHxS), and education (PFNA). For example, no meaningful associations were observed among men aged <70 years at serum collection, but among men aged ≥70 years, HRs (95% CIs) comparing Q4 to Q1 were PFHxS 1.54 (1.02–2.31) and PFOS 1.62 (1.08–2.44). No meaningful heterogeneity in associations were observed by tumor grade or stage. ConclusionsOur findings do not clearly support an association between the PFAS considered and prostate cancer. However, positive associations observed in some subgroups, and consistently positive associations observed for PFHxS warrant further investigation.

Hepatitis B Virus in People who Inject Drugs and Men who Have Sex With Men With HIV in India: A Cross-sectional Study.

People with HIV (PWH) who are coinfected with hepatitis B virus (HBV) have a higher risk of mortality compared with PWH alone. Populations such as people who inject drugs (PWID) and men who have sex with men (MSM) are particularly at high risk for HBV acquisition; yet, limited epidemiological data from these populations exist on HBV prevalence from low- and middle-income country settings (LMICs). We characterized the prevalence and correlates of HBV serological markers in a sample of PWID and MSM with HIV recruited across 15 Indian cities using hepatitis B surface antigen (HBsAg), hepatitis B core antibody (anti-HBc), and hepatitis B surface antibody (anti-HBs). Testing of stored specimens for the presence of these markers was performed on the Abbott ARCHITECT i1000 as per the manufacturer's instructions. Correlates of ever being infected with HBV (reactive for anti-HBc and/or HBsAg) and chronic HBV (reactive for HBsAg) among those ever infected were assessed using univariable and multivariable multilevel logistic regression models accounting for site-level clustering. A total of 2198 (95%) of the 2314 participants recruited for the trial were screened for HBV markers. The median age among the PWID and MSM participants was 30 and 32 years, respectively. The prevalence of ever being infected with HBV was 75.6% vs 46.9% in PWID vs MSM, respectively (P < .01); prevalence of chronic infection was also higher in PWID vs MSM (14.1% vs 9.5%; P < .01). Correlates of ever being infected with HBV among PWID included unstable housing (adjusted odds ratio [aOR], 5.02) and sharing injection paraphernalia (aOR, 2.70), and among MSM, correlates included history of injection drug use (aOR, 4.87) and gender identity. The prevalence of isolated core (anti-HBc in the absence of anti-HBs) was 34.7% vs 29.4% in PWID vs MSM (P < .05). Vaccination serostatus was <10% in both populations. In this large sample of PWID and MSM with HIV, we observed a high prevalence of serology consistent with HBV infection and low vaccination, highlighting the need for routine screening and catch-up vaccination. The high prevalence of isolated anti-HBc reactivity highlights the need to understand the risk of reactivation with this serological pattern.

Tiempos y condiciones de almacenamiento de las muestras en anatomía patológica. Recomendaciones de la Sociedad Española de Anatomía Patológica parte 1: muestras destinadas al diagnóstico

IntroductionThe correct storage of specimens in the Pathology service is of vital importance for patient safety. However, there are no clear recommendations as regarding how long samples should be stored for a minimum period. Material and methodsA working group of the Spanish Society of Anatomic Pathology has reviewed a series of recommendations established in the literature and after two rounds of consultations and a discussion and voting phase has established a series of storage time proposals. ResultsEach of the proposals is presented with the data found in the literature and sometimes offers definitions and exceptions to the proposal. ConclusionThese recommendations, which are minimums, establish a period of at least 10 years for paraffin embedded blocks (including cell blocks), histological preparations, general cytology, pathologic cervico-vaginal cytology and electron microscopy blocks; at least 3 years for cervico-vaginal cytology, 5 years for extracted nucleic acids, at least 4 weeks for tissue in formalin and from the time of diagnosis for liquid cytology material and fluids.

Assessment of long-term stored specimens in the Siriraj Hospital colorectal cancer biobank for RNA sequencing and profiling

Abstract Objectives Biobanks play an important role in advancing cancer research, yet concerns persist regarding the molecular integrity of long-term stored samples. This study assessed fresh frozen (FF) tissues and formalin-fixed paraffin-embedded (FFPE) tissues from the Siriraj Hospital colorectal cancer (CRC) biobank collected during two distinct periods (2011–2012 and 2020–2021). Methods In 2022, FF and FFPE primary cancer tissues from 75 CRC patients were evaluated. RNA sequencing (RNA-Seq) analyzed comprehensive gene expression profiles in FF tissues preserved at −80 °C, while nCounter profiling elucidated cancer-specific RNA transcripts in FFPE tissues stored at ambient temperature. Comparative analyses were conducted between specimens from 2011 to 2012 and 2020–2021. Results The FF tissues stored for approximately 10.5 years were well-suited for RNA-Seq compared to the intact tissues preserved for 1.5 years. Despite consistencies in RNA quantity, RNA integrity, amount of sequencing reads, and CRC gene signature, gene enrichment analysis revealed the decreased ribosome biogenesis, spliceosome and antifolate resistance pathways in the 2011–2012 group. Moreover, the FFPE tissues also showed no alteration in RNA quantity between the two periods, and the nCounter profiling demonstrated comparable CRC-specific gene counts in spite of the significant reduction of raw counts in the 2011–2012 group. Conclusions We report that FF tissues from CRC patients, stored for 10 years, are viable for whole transcriptome RNA-Seq, despite altered pathways such as ribosome biogenesis, spliceosome, and antifolate resistance. Moreover, 10-year-stored FFPE CRC tissues remain suitable for specific RNA profiling using the nCounter pan-cancer panel, despite a significant reduction in raw counts. These findings underscore the enduring contribution of biobanks to molecular research, highlighting their value a decade post-collection.

New insights into the DNA extraction and PCR amplification of minute ascomycetes in the genus Laboulbenia (Pezizomycotina, Laboulbeniales)

Molecular studies of fungi within the order Laboulbeniales (Ascomycota, Pezizomycotina) have been hampered for years because of their minute size, inability to grow in axenic culture, and lack of reliable and cost-efficient DNA extraction protocols. In particular, the genus Laboulbenia is notorious for low success with DNA extraction and polymerase chain reaction (PCR) amplification. This is attributed to the presence of melanin, a molecule known to inhibit PCR, in the cells. We evaluated the efficacy of a standard single cell-based DNA extraction protocol by halving the recommended amount of reagents to reduce the cost per extraction and adding bovine serum albumin (BSA) during the multiple displacement amplification step to reverse the effect of melanin. A total of 196 extractions were made, 111 of which were successful. We found that halving the reagents used in the single cell-based extraction kit did not significantly affect the probability of successful DNA extraction. Using the halved protocol reduces cost and resource consumption. Moreover, there was no significant difference in the probability of successfully extracting DNA based on whether BSA was added or not, suggesting that the amount of melanin present in cells of the thallus has no major inhibitory effect on PCR. We generated 277 sequences from five loci, but amplification of the internal transcribed spacer region, the mitochondrial small subunit rDNA, and protein-coding genes remains challenging. The probability of successfully extracting DNA from Laboulbeniales was also impacted by specimen storage methods, with material preserved in > 95% ethanol yielding higher success rates compared to material stored in 70% ethanol and dried material. We emphasize the importance of proper preservation of material and propose the design of Laboulbeniales-specific primers to overcome the problems of primer mismatches and contaminants. Our new insights apply not only to the genus Laboulbenia; Laboulbeniales generally are understudied, and the vast majority of species remain unsequenced. New and approachable molecular developments will benefit the study of Laboulbeniales, helping to elucidate the true diversity and evolutionary relationships of these peculiar microfungi.

Difference Results Bacteria Numbers with Variations Temperature and Check Time in The Elderly Urine Suspect Urinary Tract Infections (UTI)

Delays in examination have the potential to result in differences in the results of the number of bacteria in the examined specimens plus storage of specimens at non-optimal temperatures. The difference in the number of bacteria can be known by examining the Total Plate Count (TPC) in urine. The purpose of this study was to determine the differences in the results of germ numbers in terms of variations in examination delays and urine storage temperatures of elderly suspects with urinary tract infections (UTI). The type of research conducted was experimental analytic with the subject of 4 elderly urine in Kratonan Village, Surakarta with criteria sampling technique. Research data were obtained through questionnaires and examination of total plate numbers with mayo technique examination, where the results were multiplied by a conversion factor of 102 CFU/ml. The results were analyzed by SPSS statistical test using Two Way ANOVA which obtained a significant value of 0.915 (p&gt;0.05). Then continued with SPSS data analysis using Tukey Pos Hoc aims to see the real difference of each variation. There is no difference in the germ count results with temperature variations and time delays in the urine of elderly suspected urinary tract infection (UTI) patients.

Field study to determine the reliability of HIV viral load results shows minimal impact of delayed testing in South Africa.

Understanding factors that impact HIV viral load (VL) accuracy in resource-limited settings is key to quality improvement. We evaluated whether testing delay and specimen storage between 25 °C and 30 °C before testing affected results. Between November 2019 and June 2023, 249 individuals on antiretroviral therapy, or with newly diagnosed HIV, were recruited from clinics in Cape Town and Gqeberha, South Africa, and three plasma preparation tubes were collected. One tube was tested within 24 h, while the others were stored uncentrifuged at ambient temperatures before testing. Centrifugation and testing of matched samples were performed on Day 4 and Day 7 after collection. Time delay and ambient storage had minimal impact in specimens with a Day 1 VL of > 100 copies/mL. When grouped by Day 1 VL range, 96% - 100% of specimens at Day 4 and 93% - 100% at Day 7 had VLs within 0.5 log copies/mL of the first result. The greatest variability at Days 4 and 7 was observed when the Day 1 VL was < 100 copies/mL. However, there was no trend of increasing difference over time. Of Day 1 specimens with undetectable VL, or VL < 50 copies/mL, 80% had concordant results at Day 4 and 78% at Day 7. These results show that VL is stable in plasma preparation tubes for 7 days when stored at room temperature. There is significant variability in specimens with low VL, but variability is not affected by testing delay. Ideal HIV VL testing conditions are frequently unachievable in resource-limited settings. Data are needed on whether this impacts on the validity of test results. Our results provide reassurance that storage at ambient temperature for up to 7 days before testing does not substantially affect the VL result.

DNA barcoding for identification of forensically important synanthropic flesh flies (Diptera: Sarcophagidae) from Eastern India

ABSTRACT Sarcophagidae is a significant Diptera family because several of its species are among the first to visit a corpse, and it helps in minPMI estimation. Sarcophagidae species identification for a layman is difficult due to identical morphological appearance and if the fly is a female. Also, fly specimens get damaged due to improper storage of forensic specimens collected from crime scenes. Thus, in this study, we aimed to check the efficiency of cytochrome oxidase 1 (COI) for identifying eleven sarcophagid flies species. Flesh flies were collected from various biogeographic regions of West Bengal. COI barcodes successfully differentiated between species with a very significant K2P interspecific genetic divergence, which ranged between 3.12% and 20.11%, and intraspecific genetic divergences ranged from 0% to 1%. The subgenera Uroxanthisca and Sinonipponia are used in the phylogenetic analysis for the first time in the oriental context. Phylogenetic analysis was performed using a Neighbour Joining (NJ) tree constructed using Kimura two-parameter (K2P) in MEGA X software. Thus, COI barcodes proved to be quite effective as an alternate method of identifying flesh fly species, and future studies should include additional markers for further phylogenetic investigations of the Sarcophagidae family.

Comparison of the ScreenFire and Xpert HPV assays for the detection of human papillomavirus and cervical precancer among women living with HIV in Malawi

BackgroundThe World Health Organization recommends human papillomavirus (HPV) testing for primary cervical cancer screening, including among women living with HIV (WLWH). Low-and-middle-income countries account for 85% of the cervical cancer burden globally, yet have limited access to HPV-based screening, largely due to cost. This study aims to compare the performance of a rapid, isothermal amplification HPV assay (ScreenFire) to that of the Xpert HPV assay for the detection of HPV and cervical precancer among WLWH in Malawi.MethodsWe utilized stored self- and provider-collected specimens from a prospective cohort study of WLWH in Malawi from July 2020 to February 2022. Specimens were tested with both Xpert and ScreenFire HPV assays. The overall and within-channel non-hierarchical agreement between ScreenFire and Xpert was determined for both self- and provider-collected specimens. Hierarchical ScreenFire HPV positivity by channel was compared to Xpert for each histological diagnosis—cervical intraepithelial neoplasia grade 2 or worse (CIN2+) compared to <CIN2.Results315 matched self- and provider-collected specimens had valid results from both Xpert and ScreenFire testing and were included in analyses, of which 279 and 36 were HPV positive and HPV negative, respectively, on Xpert self-collection. Of the 315, 245 (78%) had normal pathology, 21 CIN1 (7%), 14 CIN2 (4%), and 35 CIN3 (11%). Of the 245 with normal pathology, 213 (87%) and 188 (77%) were HPV-positive on Xpert and ScreenFire self-collected specimens, respectively. Among provider-collected specimens, the assays had 80% agreement on overall HPV positivity (unweighted kappa 0.59, 95% 0.50–0.69). ScreenFire was HPV-positive in 90% of self-collected specimens that were HPV-positive on Xpert. Channel agreement between the assays was high for both self- and provider-collected specimens, but slightly lower for HPV18/45. In hierarchical analysis, ScreenFire demonstrated high concordance with Xpert testing for detecting CIN2+ cases in all channels, missing no HPV 16 or HPV 18/45 positive CIN2+ case that was positive on Xpert, in both self- and provider-collected specimens.ConclusionIn this study of stored specimens, the ScreenFire HPV assay performed well in the detection of HPV and CIN2+ among WLWH compared to the Xpert HPV assay. If supported by larger validation studies, ScreenFire could be an affordable alternative point-of-care HPV assay for use in LMICs.