We present a sample preparation method for cryo-electron microscopy (cryo-EM) that requires only 3–20nL of sample to prepare a cryo-EM grid, depending on the protocol used. The sample is applied and ...spread on the grid by a microcapillary. The procedure does not involve any blotting steps, and real-time monitoring allows the water film thickness to be assessed and decreased to an optimum value prior to vitrification. We demonstrate that the method is suitable for high-resolution cryo-EM and will enable alternative electron microscopy approaches, such as single-cell visual proteomics.
Abstract
Objectives
We aimed to identify potential laboratory causes of suboptimal liver biopsy quality and sought to implement corresponding measures to improve biopsy adequacy.
Methods
We ...prospectively measured the number and size of tissue fragments and the amount of portal tracts in 200 consecutive pediatric medical liver biopsies before and after quality improvement processes were initiated.
Results
We identified laboratory-related tissue fragmentation as a significant cause of low biopsy adequacy. The principal approaches to reduce fragmentation included establishment of multistep monitoring of tissue integrity, adjustment of specimen-processing conditions, and laboratory staff education and awareness. These adjustments collectively led to lower overall tissue fragmentation (decreasing from 59% to 24%, P < .01) and higher biopsy adequacy rates (increasing from 32% to 56%, P < .01). The number of evaluable portal tracts increased from 4.4 to 5.7 portal tracts per centimeter of core biopsy tissue (P < .01).
Conclusions
We demonstrated a sustainable improvement in the overall quality of pediatric needle core liver biopsies by reducing tissue fragmentation. Effective laboratory adjustments included monitoring of tissue integrity, modifications of processing conditions, and laboratory staff education.
We conducted a meta-analysis of test agreement/concordance between human papillomavirus (HPV) testing in self-collected vs clinician-collected samples in 26 studies (10 071 participants) updating a ...previous meta-analysis on accuracy for cervical precancer. Pooled overall agreement was 88.7% (95% CI: 86.3%-90.9%), positive agreement was 84.6% (95% CI: 79.9%-88.7%), negative agreement was 91.7% (95% CI: 89.1%-94.0%) and kappa was 0.72 (95% CI: 0.66-0.78). Subgroup meta-analyses suggested higher overall agreement for target amplification-based DNA assays (90.4%) compared to signal amplification-based DNA assays (86.7%; P = .175) or RNA assays (82.3%; P < .001). HPV test agreement/concordance targets may provide criteria to extend existing validations toward alternative sampling approaches and devices/storage media.
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.
Although millions of transfusions are given annually worldwide, the effect of red blood cell (RBC) unit storage duration on oxygen delivery is uncertain.
To determine if longer-storage RBC units are ...not inferior to shorter-storage RBC units for tissue oxygenation as measured by reduction in blood lactate levels and improvement in cerebral tissue oxygen saturation among children with severe anemia.
Randomized noninferiority trial of 290 children (aged 6-60 months), most with malaria or sickle cell disease, presenting February 2013 through May 2015 to a university-affiliated national referral hospital in Kampala, Uganda, with a hemoglobin level of 5 g/dL or lower and a lactate level of 5 mmol/L or higher.
Patients were randomly assigned to receive RBC units stored 25 to 35 days (longer-storage group; n = 145) vs 1 to 10 days (shorter-storage group; n = 145). All units were leukoreduced prior to storage. All patients received 10 mL/kg of RBCs during hours 0 through 2 and, if indicated per protocol, an additional 10 mL/kg during hours 4 through 6.
The primary outcome was the proportion of patients with a lactate level of 3 mmol/L or lower at 8 hours using a margin of noninferiority equal to an absolute difference of 25%. Secondary measures included noninvasive cerebral tissue oxygen saturation during the first transfusion, clinical and laboratory changes up to 24 hours, and survival and health at 30 days after transfusion. Adverse events were monitored up to 24 hours.
In the total population of 290 children, the mean (SD) presenting hemoglobin level was 3.7 g/dL (1.3) and mean lactate level was 9.3 mmol/L (3.4). Median (interquartile range) RBC unit storage was 8 days (7-9) for shorter storage vs 32 days (30-34) for longer storage without overlap. The proportion achieving the primary end point was 0.61 (95% CI, 0.52 to 0.69) in the longer-storage group vs 0.58 (95% CI, 0.49 to 0.66) in the shorter-storage group (between-group difference, 0.03 95% CI, -0.07 to ∞, P < .001), meeting the prespecified margin of noninferiority. Mean lactate levels were not statistically different between the 2 groups at 0, 2, 4, 6, 8, or 24 hours. Kaplan-Meier analysis and global nonlinear regression revealed no statistical difference in lactate reduction between the 2 groups. Clinical assessment, cerebral oxygen saturation, electrolyte abnormalities, adverse events, survival, and 30-day recovery were also not significantly different between the groups.
Among children with lactic acidosis due to severe anemia, transfusion of longer-storage compared with shorter-storage RBC units did not result in inferior reduction of elevated blood lactate levels. These findings have relevance regarding the efficacy of stored RBC transfusion for patients with critical tissue hypoxia and lactic acidosis due to anemia.
clinicaltrials.gov Identifier: NCT01586923.
In the setting of supply chain shortages of nasopharyngeal (NP) swabs, we sought to compare the ability of nasopharyngeal, midturbinate nasal, and oropharyngeal swabs (NPS, MTS, and OPS) to detect ...SARS-CoV-2. Community and hospitalized participants post-COVID-19 diagnosis were swabbed and tested for SARS-CoV-2 by PCR. Thirty-six participants had all 3 swabs collected. Using detection at any site as the standard, the percent positive agreements were 90% (95% CI 74.4−96.5), 80% (70.3−94.7) and 87% (62.7−90.5) for NPS, MTS, and OPS, respectively. Subsequently, 43 participants had OPS and NPS collected. Thirty-nine were positive with a percent positive agreement of 82.1% (95% CI 67.3−91.0) for OPS and 87.2% (73.3−94.4) for NPS. Combining all 79 patients tested, 67 were positive at either site with a positive agreement was 86.5% (76.4−92.7) for OPS and 91.1% (81.8−95.8) for NPS. OPS are an acceptable alternative to NPS for the detection of SARS-CoV-2 infections.
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.
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.
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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